JP2007537276A5 - - Google Patents

Description

Method of inhibiting pro-inflammatory cytokine expression using ghrelin

(Background of the Invention)
Inflammation is a complex stereotypic response of the body that represents a response to cellular and vascular tissue damage. The discovery of detailed processes of inflammation has revealed a close relationship between inflammation and immune responses. The main features of the inflammatory response are vasodilation (ie, the blood vessels expanding to increase blood flow to the psoriatic area); vascular permeability (allowing diffusible components to enter the site); Cellular invasion due to chemotaxis, or directional migration of inflammatory cells through the walls of blood vessels to the site of injury; changes in biosynthetic, metabolic and catabolic profiles of many organs; and activation of cells of the immune system and It is the activation of a complex enzyme system in plasma.

There are two forms of inflammation (acute and chronic). Acute inflammation can be divided into several stages. The initial overall event of the inflammatory response is temporary vasoconstriction (ie, stenosis of the blood vessels caused by contraction of the smooth muscles of the vascular wall), which can be seen as skin blanching. This is followed by several stages that occur after minutes, hours and days. The first is an acute vascular reaction, which lasts within a few seconds of tissue damage and lasts for a few minutes. This results in vasodilation and increased capillary permeability due to changes in the vascular endothelium, which leads to increased blood flow (erythema) that causes redness (hyperemia) and fluid entry into the tissue (edema). .

  An acute vascular response can follow an acute cellular response, which occurs in the next few hours. A characteristic of this stage is the appearance of granulocytes (especially neutrophils) in the tissue. These cells first attach themselves to endothelial cells in the blood vessels (marginal orientation) and then enter the surrounding tissue (leakage). During this phase, red blood cells can also leak into the tissue and cause bleeding. When a blood vessel is damaged, fibrinogen and fibronectin are deposited at the site of the injury, platelets aggregate and activate, red blood cells overlap with what is called “remuneration”, helping to stop bleeding, clot formation Help. Dead and dying cells contribute to pus formation. If the damage is severe enough, a chronic cellular response can continue over the next few days. A characteristic of this stage of inflammation is the appearance of mononuclear cell infiltration consisting of macrophages and lymphocytes. Macrophages are involved in microbial death, removal of cellular and tissue debris from cells, and tissue remodeling.

  Chronic inflammation is a long-term (weeks, months, or even indefinite) inflammatory response whose long time course is caused by the persistence of the causative stimulus for tissue inflammation. This inflammatory process inevitably causes tissue damage and involves simultaneous attempts of healing and repair. The exact nature, extent and time course of chronic inflammation is variable and depends on the balance between the causative factor and the body's attempt to remove it. The etiological factors that cause chronic inflammation include the following. (I) Infectious organisms that avoid or resist host defense and are therefore present in tissues for a long time (human tuberculosis, actinomycetes, and many fungi, protozoa, and metazoan parasites Can be mentioned). Such organisms generally can avoid phagocytosis or survive in phagocytic cells and produce little toxin that causes acute tissue damage. (Ii) Infectious organisms present in damaged areas that are not inherently resistant but are protected from host defense. An example is a bacterium that grows with pus in an abscess cavity that is not drained, which is protected from both host immunity and blood-borne therapeutic factors (eg, antibiotics). Some locations are particularly prone to chronic abscesses (eg, bone and pleural space). (Iii) Irritating non-viable foreign bodies that cannot be removed by enzymatic degradation or phagocytosis. Examples include a wide range of materials implanted in wounds (wood thorns, grit, metal and plastic), a wide range of inhaled materials (silica dust and other particles or fibers), or a wide range of materials that are deliberately introduced (Surgical prostheses, sutures, etc.). Also included are grafts. Dead tissue components that cannot be degraded can have a similar effect (eg, keratin scales derived from fragments of ruptured epidermoid cysts or dead bones of osteomyelitis (separated pieces)). (Iv) In some cases, the stimulus to chronic inflammation may be a normal tissue component. This occurs with inflammatory diseases (ie, autoimmune diseases) where the disease process is initiated and maintained due to dysregulation of the immune response to the body's own tissues. This response is seen in older and older subjects. (V) For many diseases characterized by chronic inflammatory pathological processes, the root cause remains unknown. An example is Crohn's disease (Figure 11).

  Inflammation and innate immune activation are common responses to non-replicating adenoviruses (Ad) used as vectors for gene therapy (Non-Patent Document 1; Non-Patent Document 2). The complement system is central to innate immunity and inflammation (Non-Patent Document 3). Since the complement system consists of multiple membrane-bound and blood factors, this complement system is particularly relevant for the delivery of intravenously administered vectors. Indeed, Non-Patent Document 4 shows that complement was activated in the majority of human plasma samples when challenged with different adenovirus serotypes; complement activation was completely dependent on anti-Ad antibodies. (Cichon (2001)).

  Complement-mediated inactivation is a multi-step enzymatic cascade that ultimately results in the formation of a cell membrane injury complex (MAC) that mediates cell membrane penetration and subsequent lysis of invading organisms. This is triggered either by an antigen-antibody complex (classical pathway) or via an antibody-independent pathway (alternate pathway) activated by certain polysaccharides, viruses and bacteria. .

  Human organs and cells are themselves protected against complement-mediated lysis. This protection is achieved by the expression of complement inactivators. Until now. Five human factors are known. CD35 (CR1) is released from the cell and acts primarily exogenously. In contrast, CD59, CD46 (MCP), CD55 (DAF) and HRF are incorporated into the cell membrane. CD46 (MCP) is a classic transmembrane protein, whereas HRF, CD59 and CD55 are anchored to GPI. These factors can interfere with the complement cascade at two different stages: DAF, CR1 and MCP act at the early stages of both the alternative and classical pathways. In contrast, CD59 and HRF inhibit assembly of the cell membrane injury complex, the final step in both pathways that result in channel formation and lysis.

  The initial pro-inflammatory cascade can be triggered by endotoxins (also known as lipopolysaccharides or LPS). LPS is one of the main constituents of the cell wall of Gram-negative bacteria. Recognition of conserved microbial products (eg, LPS) by the innate immune system results in a variety of signaling pathways. These signaling pathways mediate the induction and secretion of cytokines that can regulate the level and duration of the inflammatory response. The systemic inflammatory response with endotoxic shock (caused by triggers such as the presence of LPS) is controlled by the levels of pro- and anti-inflammatory cytokines. Pro-inflammatory cytokine production by cells of the innate immune system plays an important role in mediating early host defense against invading pathogens (5) and regulates the nature or duration of host inflammatory responses Inability to do so can often mediate harmful host effects such as those observed in chronic and inflammatory diseases. Furthermore, in the early stages of sepsis, the host inflammatory response is thought to be in an over-activated state with a dominant increase in the production of pro-inflammatory cytokines that mediate host tissue damage and lethal shock (non- Patent Document 6). In this regard, the ability to suppress pro-inflammatory cytokines and / or enhance anti-inflammatory cytokines (ie IL-10) has been shown to greatly reduce the toxic effects of endotoxins (Non-Patent Document 7). Non-patent document 8).

Ghrelin is a polypeptide in which 28 amino acids secreted mainly from gastric X / A-like cells are acylated (Non-patent Document 9). Ghrelin is involved in long-term regulation of growth hormone (GH) release, energy balance, food intake, and body weight in rodents (10, 11) and humans (12). . The ghrelin gene encodes a 117 amino acid peptide, which is pre-pro-ghrelin that shares 82% homology between rat and human (9). ). Ghrelin is considered to be the only known circulating appetite promoting factor and exerts an antagonistic effect on the leptin-induced decrease in food intake through activation of the hypothalamic NPY / Y1 pathway (Non-Patent Document 11, Non-Patent Document 11) Reference 13). The effect of ghrelin is mediated through seven transmembrane G protein-coupled receptors called growth hormone secretagogue receptor GHS-R (Non-patent Document 14). This receptor is evolutionarily conserved from puffer fish to humans (15), indicating that ghrelin plays a fundamental role in the growth and development of organisms. The GH S -R1a type receptor is involved in GH release, and an unspliced non-functional receptor mRNA variant identified as GHS-R 1b has been identified in a wide range of tissues including lymphoid organs. (Non-Patent Document 16). Hexarelin is a synthetic analog that binds to GHS-R and induces GH secretion from porcine and bovine peripheral blood mononuclear cells (PBMC), which indicates that the GHS-R ligand is somehow directly into the immune system. (Non-patent Document 17). Furthermore, the extensive tissue distribution of GHS-R in the lymphatic system suggests that ghrelin and GHS-R ligands can function as signal regulators between the endocrine, nervous and immune systems.

Inflammatory cytokines released from immune cells have been shown to act in the central nervous system (CNS) to control food intake and energy homeostasis (18). Reduced food intake or loss of appetite is one of the most common symptoms of illness, injury or inflammation (19). Cytokines such as IL-1β, IL-6, and TNF-α are associated with inflammation associated with inflammation (Non-Patent Document 20), chronic mild inflammation in aging (Non-Patent Document 21, Non-Patent Document 22) and Involved in atherosclerosis (Non-patent Document 23). What is needed in the art is to regulate inflammatory cytokine production by exogenous factors such as ghrelin to ameliorate a wide range of diseases and disease states.
Jooss, K.M. Gene Ther. , 2003, 10, p. 955-963 Zaiss, A .; K. J. et al. Virol. , 2002, 76, p. 4580-4590 Walport, M.M. J. et al. N Eng J Med, 2001, 344, p. 1058-1066 and 1140-1144 Cicho et al., Gene Ther, 2001, 8, p. 1794-1800 O'Neill LA, Dinarello CA. The IL-1 receptor / toll-like receptor superfamily: clinical receptors for inflation and host defense. , Immunol Today. , 2000, 21 (5), p. 206-9 Cohen, J The immunopathogenesis of sepsis. , Nature, 2003, 420, p. 885-891 Berg DJ, Kuhn R, Rajewsky K, Muller W, Menon S, Davidson N, Grunig G, Rennick D. et al. Interleukin-10 is a central regulator of the response to LPS in murine models of the endotoxic shock and the Swartzman reaction buttonot. , J Clin Invest. , November 1995, 96 (5), p. 2339-47 Howard M, Muchamuel T, Andrade S, Menon S. , Interleukin 10 protects rice from endotoxemia. , J Exp Med. Apr. 1, 1993, 177 (4), p. 1205-8 Kojima et al., Nature. 1999, 402, p. 656-660 Tschop et al., Nature, 2000, 407, p. 908-913 Nakazato et al., Nature. 2001, 409, p. 194-198 Cummings et al., New Engl. J. et al. Med. , 2002, 346, p. 1623-1630 Inui, A .; Ghrellin Nature Rev. Neurosci. , 2001, 2, p. 551-560 Howard et al., Science. 1996, 273, p. 974-977 Paryha et al., Mol. Endocrinol. 2000, 14, p. 160-169 Gnanapavan et al. Clin. Endocrinol. Metab. , 2002, 87, p. 2988-2991 Dantzer, R.A. Ann. NY. Acad. Sci. 2001, 933, p. 222-234 Hart, BL. Neurosci. Biobehav. Rev. 1998, 12, p. 123-137 Kotler, D .; P. Ann. Internal Med. 2000, 133, p. 622-634 Ershler et al., Annu. Rev. Med. 2000, 51, p. 245-270 Bruunsgaard et al., Curr. Opin. Hematol. 2001, 8, p. 131-136 McCarty, M.M. F. Med. Hypotheses, 1999, 52, p. 465-477 Bochkov et al., Nature. , 2002, 419, p. 77-81

(Summary of the Invention)
The present invention provides a method of treating inflammation comprising administering ghrelin or a fragment thereof.

  The invention also provides a method of treating loss of appetite comprising administering ghrelin or a fragment thereof.

  The present invention also provides a method of treating sepsis comprising administering ghrelin or a fragment thereof.

  The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and, together with the description, illustrate the principles of the invention. Help explain.

(Detailed description of the invention)
The present invention refers to the following detailed description of the preferred embodiments of the invention, and to the examples included herein, and to the drawings and the foregoing description and the following description. Can be more easily understood.

(A. Definition)
Before the method and composition are disclosed and described, the invention is not limited to a particular method or material, unless otherwise specified, and is not limited to a particular reagent unless otherwise specified. It is understood that it will not. Of course, because these can fluctuate. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

  As used herein and in the appended claims, the singular forms “a”, “an”, and “the” clearly indicate the context otherwise. Unless otherwise stated, includes multiple references. Thus, for example, reference to “a substance” includes one or more substances, and the like.

Ranges may be expressed herein as from “about” a particular value and / or to “about” a particular value. When ranges are expressed in this way, other embodiments include from the one particular value and / or to the other particular value. Similarly, where a value is expressed as an approximation, it is understood that the use of the antecedent “about” forms that particular value forms another embodiment. It is further understood that the endpoints of each range are important both for the other endpoint and the independence of the other endpoint .
The terms “higher”, “increases”, “elevates” or “elevation” increase above basal levels or increase when compared to controls. Say. The terms “low”, “lower”, “inhibits”, “inhibition”, “reduces” or “reduction” are less than the basal level. Decrease down or decrease when compared to control. For example, the basal level before inflammation or the addition of an agent that causes inflammation, or in the absence or presence of an agent that causes inflammation, is a normal level in vivo.

  The terms “mediate” or “mediation” and “modulate” or “modulation” mean to regulate or regulate, in particular to increase or enhance. , Increase or decrease, inhibit, or the terms “mediate” and “modulate” are used interchangeably throughout.

  “Inflammation” or “inflammatory” is defined as the response of a living tissue to injury, infection or exposure. Anything that stimulates an inflammatory response is said to be inflammatory.

  “Inflammatory disease” is defined as any disease state associated with inflammation.

  An “infection” or “infection process” is defined as an organism being invaded by any type of foreign material or other organism. The consequences of infection may include growth of the foreign organism, production of toxins, and damage to the host organism. Examples of infection include viral infection, bacterial infection, parasitic infection, and fungal infection.

  “Liver toxicity” is defined as an abnormal accumulation of toxic substances in the liver. A number of diagnostic criteria can be used to assess the medical significance of the following toxicity data: (a) injury type / severity, (b) reversibility, (c) characteristic mechanism, (d ) Differences between species, (e) availability of susceptibility markers of toxicity, (e) safety margin (non-toxic dose / pharmacologically active dose), and (f) therapeutic potential.

  “Cancer therapy” is defined as any treatment or therapy useful for preventing, treating or ameliorating symptoms associated with cancer. Cancer treatment methods can include, but are not limited to, apoptosis induction, radiation therapy, and chemotherapy.

  “Transplant” is defined as the transplantation of an organ or body part from one organism to another.

  “Transplant rejection” is defined as an immune response elicited by the presence of foreign blood or tissue in a subject's body. In one example of transplant rejection, antibodies are formed against foreign antigens in the transplanted material.

  As used throughout, an “individual” means an individual. Accordingly, this “subject” includes domestic animals (eg, cats, dogs, etc.), livestock (eg, cows, horses, pigs, sheep, goats, etc.), laboratory animals (eg, mice, rabbits, rats, etc.). , Guinea pigs, etc.), and birds. Preferably the subject is a primate and more preferably a human.

  The term “control level” or “control cell” is defined as the standard by which changes are measured, for example, these controls are not subjected to experiments, but instead are subjected to a defined set of parameters, or these Control is based on pre-treatment level or post-treatment level.

  By “treating” is meant that an improvement in disease state (ie, inflammatory response) is observed and / or detected upon administration of a substance of the invention to a subject. Treatment can range from a positive change in disease symptom (s) to complete recovery of the inflammatory response as detected by techniques known in the art (eg, disease severity or Reduced intensity, changes in medical parameters indicating the condition of the subject, relief of pain or increased or increased function).

  By “preventing” is meant that after administration of a substance of the invention to a subject, the subject does not develop inflammatory symptoms.

(B. Composition)
Disclosed are the components used to prepare the disclosed compositions and the compositions themselves. When these substances and other substances are disclosed herein and combinations of these substances, subsets of these substances, interactions of these substances, groups of these substances, etc. are disclosed, It is understood that each is specifically contemplated and disclosed herein, even though specific combinations and permutations and specific references to each of the collective combinations and permutations may not be explicitly disclosed. For example, if a particular peptide is disclosed and discussed, and many modifications that can be made to many molecules that contain the peptide are considered, modifications with amino acids within the peptide (unless indicated otherwise) Each combination and permutation with each other) and all combinations and permutations thereof are specifically contemplated. Thus, when the classes of molecules A, B and C and the classes of molecules D, E and F are disclosed, and combined molecule AD examples are disclosed, each is individually described even though each is not individually described. And AE, AF, BD, BE, BF, CD, CE, and CF are disclosed. It is regarded. Similarly, any subset or combination of these is also disclosed. Thus, for example, the sub-group of AE, BF, and CE is considered disclosed. This concept applies to all aspects of the present application, including, but not limited to, steps in methods of making and using these disclosed compositions. Thus, where there are additional various steps that can be performed, each of these additional steps is performed by any particular embodiment of the disclosed method or any of the disclosed method embodiments. It can be implemented by specific combinations.

(1. Ghrelin)
The term “ghrelin” is used throughout to refer to any ghrelin molecule disclosed above or a functional fragment thereof. The invention includes a method of treating inflammation in a subject, the method comprising administering to the subject an effective amount of SEQ ID NO: 1 or a fragment thereof. SEQ ID NO: 1 represents full-length ghrelin (Accession No. AB029434). Also contemplated is a method of treating inflammation in a subject, comprising: an effective amount of SEQ ID NO: 3 (amino acids 1-18 of full length ghrelin), or SEQ ID NO: 4 (amino acids 1 to 1 of full length ghrelin). 14) or an effective amount of SEQ ID NO: 5 (amino acids 1 to 10 of full-length ghrelin) of the fragment or an effective amount of SEQ ID NO: 6 (amino acids 1 to 5 of full-length ghrelin) of the fragment The step of administering to. Also contemplated is administering a fragment of any length of the above sequence that is a functional ghrelin molecule.

  Ghrelin specifically inhibits the expression and production of inflammatory cytokines (eg IL-1β, IL-6 and TNF-α) by human PBMC and T cells via functional cell surface GHS-R Work both effects and selective inhibitory effects. GHS-R in primary and cultured human T cells, like other classical GPCRs, causes a strong intracellular calcium release upon ligation with its natural ligand, ghrelin, and Preferentially associates with GM1 lipid rafts upon activation within the cell. Consistent with the expression of functional GHS-R in T cells, ghrelin actively induces actin polymerization in T cells. Like chemokine (SDF-1), ghrelin treatment leads to cellular polarization of leukocytes and from linear cortical morphology with dormant lymphocytes, at the tip and contact areas with polarized activated T cells. It leads to a change in the distribution of actin to a more concentrated form (Taub et al., Science. 260: 355-358 (1993), Inui, A Cancer Res. 59: 4493-4501 (1999)). These GPCR-like redistribution patterns represent an important role for GHS-R in immune cell signaling and transport.

Previously, ghrelin was only thought to be produced by gastric endocrine-like cells and then released into the circulation. Many analytical techniques have demonstrated that ghrelin is endogenously produced and secreted by both T cells and PBMC in a manner similar to many immune-derived cytokines. It has been found that most T cells tested from human donors constitutively express low levels of endogenous ghrelin and are significantly increased upon cell activation. Activated T cells express and produce ghrelin protein. This indicates that the pre-propeptide must be actively cleaved within the T cell to yield an active ghrelin peptide. Like some cytokines (eg, TGF-β) and hormones (eg, TSH), these precursor proteins are synthesized and subsequently stored for rapid cleavage and use when needed. Furthermore, the expression and secretion of mature ghrelin from T cells after activation via a T cell receptor ligand has been demonstrated. In human subjects, gastrectomy results in only a 35-50% reduction in circulating ghrelin, and ghrelin levels increase to 2/3 of pre-gastrectomy levels. It has been shown that other tissues compensate to maintain that ghrelin (Hosoda H et al., J Biol Chem. 2003 Jan 3; 278 (1): 64-70). Secretion of ghrelin from T cells indicates that immune cell-derived ghrelin constitutes part of the remaining concentration of circulating ghrelin. In addition, ghrelin is also a mere known hormone that the hydroxyl group of the third serine residue is acetylated by n-octanoic acid, and this acetylation is important to some of the biological activity of the polypeptide. It is considered (Kojima et al. (1999)). N-terminal acetylated peptides are known to preferentially aggregate in cholesterol-rich microregions (Basa et al., Neurosci. Lett. 343: 25-28 (2003)) and ghrelin is activated T It is immunoreactive in cells and coexists highly within the GM1 ⁺ region rich in cholesterol. These results indicate that ghrelin is specifically targeted to the plasma membrane, promotes its interaction with its own transmembrane receptor, and optimally mediates receptor-ligand interactions. Such pathways indicate a role for ghrelin in the regulation of immune responses. Furthermore, the localization of ghrelin production plays an important role in the rapid control of ongoing and leptin-mediated responses within the local microenvironment.

(2. Homology / Identity)
One known method for defining any known variants and derivatives of the genes and disclosed proteins, or possible variants and derivatives, disclosed herein is their homology to specific known sequences. It is understood that via defining variants and derivatives. The term “ghrelin” is used throughout to refer to any ghrelin molecule or functional fragment thereof. A “fragment” is defined as any subportion of a reference sequence. For example, SEQ ID NO: 2 represents the specific sequence of a nucleic acid molecule encoding ghrelin, and SEQ ID NO: 1 represents the specific sequence of the protein encoded by SEQ ID NO: 2 (ghrelin protein). The method of the invention comprises using full-length ghrelin represented by SEQ ID NO: 1 (GenBank accession number AB029434), and fragments thereof. Also included are sequences longer than SEQ ID NO: 1 and include amino acids before and / or after the functional ghrelin molecule. Examples of fragments of SEQ ID NO: 1 and sequences longer than functional molecules of SEQ ID NO: 1 that are useful in the methods disclosed herein include: amino acids 1-5 (shown in SEQ ID NO: 6) Amino acids 1-6, amino acids 1-7, amino acids 1-8, amino acids 1-9, amino acids 1-10 (shown in SEQ ID NO: 5), amino acids 1-11, amino acids 1-12, amino acids 1-13 , Amino acids 1-14 (shown in SEQ ID NO: 4), amino acids 1-15, amino acids 1-16, amino acids 1-17, amino acids 1-18 (shown in SEQ ID NO: 3), amino acids 1-19, amino acids 1- 20, amino acids 1-21, amino acids 1-22, amino acids 1-23, amino acids 1-24, amino acids 1-25, amino acids 1-26, amino acids 1-27, amino acids 1-28, amino acids 1- 9, amino acid 1-30, amino acid 1-31, amino acid 1-32, amino acid 1-33, amino acid 1-34, amino acid 1-35, amino acid 1-36, amino acid 1-37, amino acid 1-38, amino acid 1- 39, amino acid 1-40, amino acid 1-41, amino acid 1-42, amino acid 1-43, amino acid 1-44, amino acid 1-45, amino acid 1-46, amino acid 1-47, amino acid 1-48, amino acid 1- 49, amino acids 1-50, amino acids 1-75, amino acids 1-100, amino acids 1-125, amino acids 1-150, amino acids 1-175, amino acids 1-200, amino acids 1-225, amino acids 1-250, amino acids 1- 300, amino acids 1-350, amino acids 1-400, amino acids 1-450, and amino acids 1-500, and between these Fragments of all lengths.

Also specifically disclosed are these and other genes and protein variants herein, which are at least 70%, 71%, 72%, 73%, 74%, 75% to the presented sequence. %, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, It has 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % homology. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids (eg, genes). For example, after aligning the two sequences so that the homology is at its highest level, the homology can be calculated.

Another way of calculating homology can be performed by known algorithms. Optimal sequence alignment for comparison is described by Smith and Waterman Adv. Appl. Math. 2: 482 (1981), local homology algorithm, Needleman and Wunsch (J. Mol Biol. 48: 443 (1970)), Pearson and Lipman (Proc. Natl. Acad. Sci. US A.85: 2444 (1988)) similarity search method, Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, Wis.) And computer implementations of these algorithms (GAP, BESTFAIT, FASTA, FASTAST) Or by visual inspection.

  The same type of homology is described, for example, in Zuker, M .; Science 244: 48-52, (1989), Jaeger et al. Proc. Natl. Acad. Sci. USA 86: 7706-7710 (1989), Jaeger et al. Methods Enzymol. 183: 281-306 (1989), which can be obtained for nucleic acids by the algorithms disclosed in at least materials related to nucleic acid alignment, incorporated herein by reference.

(3. Nucleic acid)
Various nucleic acid-based molecules are disclosed herein, including, for example, nucleic acids, and various functional nucleic acids that encode, for example, ghrelin and any other protein disclosed herein. The disclosed nucleic acids are composed of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. For example, it is understood that when a vector is expressed in a cell, the expressed mRNA is typically composed of A, C, G and U. Similarly, for example, when an antisense molecule is introduced into a cellular environment, eg, via exogenous delivery, the antisense molecule is composed of nucleotide analogs that reduce degradation of the antisense molecule in the intracellular environment. It is understood that it is advantageous.

(A. Nucleotides and related molecules)
A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through a phosphate moiety and a sugar moiety to form a linkage of nucleotides. The base portion of the nucleotide comprises adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T ). The sugar moiety of the nucleotide is ribose or deoxyribose. The phosphate portion of the nucleotide is a pentavalent phosphate. Non-limiting examples of nucleotides are 3′-AMP (3′-adenosine monophosphate) or 5′-GMP (5′-guanosine monophosphate).

  Nucleotide analogs are nucleotides that contain some type of modification either in the base moiety, sugar moiety or phosphate moiety. Modifications to nucleotides are known in the art and include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, and sugar moieties or phosphorus Modifications in the acid moiety are included.

  Nucleotide substitutes are molecules that have functional properties similar to nucleotides but do not contain a phosphate moiety, such as peptide nucleic acids (PNA). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked to each other through moieties other than the phosphate moiety. Nucleotide substitutes can adapt to a double helix-like structure when interacting with the appropriate target nucleic acid.

  It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs, for example to enhance cellular uptake. The conjugate can be scientifically linked to a nucleotide or nucleotide analog. Such conjugates include, but are not limited to, lipid moieties (eg, cholesterol moieties) (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86, 6553-6556 (1989)).

  A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog or nucleotide substitute. The Watson-Crick face of nucleotides, nucleotide analogs or nucleotide substitutes includes purine base nucleotides, nucleotide analogs or nucleotide substitutes C2, N1 and C6 positions, and pyrimidine base nucleotides, nucleotide analogs or nucleotide substitutes. Examples include C2, N3 and C4 positions.

  Hoogsteen interactions are interactions that occur on the Hoogsteen face of nucleotides or nucleotide analogs, which are exposed in the major groove of double-stranded DNA. The Hoogsteen surface includes reactive groups (NH2 or O) at the N7 position of purine nucleotides and the C6 position of purine nucleotides.

(B. Array)
For example, there are various sequences for ghrelin and other proteins disclosed herein, which are disclosed in Genbank, and these and other sequences are herein described in their entirety and in their entirety. The individual partial sequences contained in are incorporated by reference.

  Various sequences are provided herein, and these and others can be found in Genbank (www.pubmed.gov). Those of skill in the art understand how to resolve sequence mismatches and differences, and how to apply compositions and methods related to a particular sequence to other related sequences. Primers and / or probes can be designed for any sequence given the information disclosed herein and known in the art.

(4. Peptides)
(A. Peptide variant)
As discussed herein, there are many variants of ghrelin protein that are known and contemplated herein. In addition, derivatives of ghrelin protein exist for known functional ghrelin species variants, and these derivatives also function in the disclosed methods and compositions. Protein variants or derivatives are well known to those of skill in the art and may include amino acid sequence variants. For example, the amino acid sequence can typically be divided into one or more of three classes: substitutional variants, insertion variants, and deletion variants. Insertions include amino terminal fusions and / or carboxyl terminal fusions as well as internal sequence insertions of one or more amino acid residues. Insertions are usually fewer insertions than amino-terminal or carboxyl-terminal fusions, for example on the order of 1-4 residues. The immunogen can be obtained by fusing a polypeptide large enough to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Sex fusion protein derivatives (eg, those described in the Examples) are made. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, up to about 2-6 residues are deleted at any one site in the protein molecule. These variants are usually prepared by site-directed mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, which is then expressed in recombinant cell culture. Techniques for creating substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of a single residue, but can occur simultaneously at multiple different sites; insertions are usually on the order of about 1-10 amino acid residues; and deletions are approximately 1-30 residues It extends to the group. Deletions or insertions are preferably made in adjacent pairs (ie, 2-residue deletions or 2-residue insertions). Substitutions, deletions, insertions or any combination thereof can be combined to arrive at the final construct. These mutagenesis must not be set to sequences outside the reading frame. And preferably, it does not form a complementary region capable of producing the secondary structure of mRNA. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions are generally made according to Tables 1 and 2 below and are referred to as conservative substitutions.

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.

  Substantial changes in function or immunological identity are achieved by selecting substitutions that are less conservative than those in Table 2, ie, selecting residues that are more significantly different in their effect of maintaining: Generated: (a) structure of the peptide backbone within the substitution region, (b) charge or hydrophobicity of the molecule at the target site, or (c) bulkiness of the side chain. In general, substitutions that are expected to produce the greatest change in protein properties are: (a) hydrophilic residues (eg, seryl or threonyl residues) are hydrophobic residues (eg, leucyl residues, isoleucyl residues). A group substituted (or by a hydrophobic residue) instead of (group, phenylalanyl residue, valyl residue, or alanyl residue); (b) cysteine or proline in place of any other residue A substitution that is substituted (or by any other residue); (c) a side that has a positively charged side chain (eg, a lysyl, arginyl, or histidyl residue) that is negatively charged. Substitutions substituted for residues with chains (eg glutamyl or aspartyl residues) (or residues with negatively charged side chains); or (d) bulky side chains A substitution in which a residue (eg, phenylalanine) to be substituted (or in this case glycine) instead of (or by a residue having no side chain), (e) sulfation And / or substitution by increasing the number of sites for glycosylation.

  For example, replacing one amino acid with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution is the replacement of one hydrophobic residue with another or the replacement of one polar residue with another. Examples of the conservative substitution include Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each explicitly disclosed sequence are encompassed within the mosaic polypeptides provided herein.

  Substitution mutagenesis or deletion mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletion of cysteine or other unstable residues may also be desirable. Deletion or substitution of a potential proteolytic site (eg, Arg) is, for example, by deleting one of the basic residues or replacing the residue with a glutaminyl or histidyl residue Can be achieved.

  Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl or asparaginyl residues are often deaminated post-translationally to the corresponding glutamyl or asparyl residue. Alternatively, these residues are deaminated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of o-amino groups of lysine side chains, arginine side chains and histidine side chains (T E. Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco pp 79-86 (1983)), N-terminal amine acetylation, and in some cases C-terminal carboxyl amidation .

  It will be appreciated that one method of defining variants or derivatives of the proteins disclosed herein is by defining the variants and derivatives with respect to homology / identity to specific known sequences. For example, SEQ ID NOs: 1, 3, 4, 5 and 6 describe specific sequences for ghrelin. These proteins and other protein variants disclosed herein are specifically disclosed, and these variants are at least 50%, 51%, 52%, 53% relative to the presented sequence. 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70 %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, Has a homology of 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Those skilled in the art readily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at the highest level.

  Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison is either by Smith and Waterman's local homology algorithm (Adv. Appl. Math. 2: 482 (1981)) or by Needleman and Wunsch's homology alignment algorithm (J. Mol Biol. 48). : 443 (1970)) or by Pearson and Lipman's similarity search method (Proc. Natl. Acad. Sci. USA 85: 2444 (1988)) or computer implementation of these algorithms ( GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science D r., Madison, WI) or by visual inspection.

  A similar type of homology for nucleic acids is described, for example, in Zuker, M .; Science 244: 48-52 (1989), Jaeger et al. Proc. Natl. Acad. Sci. USA 86: 7706-7710 (1989), Jaeger et al. Methods Enzymol. 183: 281-306 (1989), which are incorporated herein by reference for at least materials for nucleic acid alignment.

  Conservative mutagenesis and homology descriptions are combined together in any combination (eg, in embodiments having at least 70% homology to a particular sequence when the variant is conservatively substituted). It is understood that you get.

  Since this specification discusses various proteins and various protein sequences, it is understood that the nucleic acids encoding those protein sequences are also disclosed. This includes all degenerate sequences for a particular protein sequence, ie, nucleic acids having a sequence encoding one particular protein, as well as all nucleic acids encoding the disclosed variants and derivatives of that protein sequence (degenerate). Nucleic acid). Thus, even though each specific nucleic acid sequence cannot be noted herein, each and every sequence is actually disclosed and described herein via its disclosed protein sequence. Understood. For example, one of many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 1 is set forth in SEQ ID NO: 2. The amino acid sequence does not indicate which particular DNA sequence in the organism encodes the protein (wherein certain variants of the disclosed protein are disclosed herein) It is understood that known nucleic acid sequences that encode that protein in the particular sequence that is generated are also known and are disclosed and described herein.

  It is understood that there are many amino acid and peptide analogs that can be incorporated into the disclosed compositions. For example, there are D amino acids or amino acids having different functional substituents other than the amino acids shown in Tables 1 and 2. Reverse stereoisomers of naturally occurring peptides and stereoisomers of peptide analogs are disclosed. These amino acids can be modified in a polypeptide by altering the tRNA molecule by amino acid selection and by manipulating genetic constructs that insert amino acid analogs into the peptide chain in a site-specific manner, for example using amber codons. (Thorson et al., Methods in Molec. Biol. 77: 43-73 (1991), Zoller, Current Opinion in Biotechnology, 3: 348-354 (1992); Iba, Biotechnology & Genetology & Genetology & Genetology & Genetology & Genetology & Genetology 197-216 (1995), Chill et al., TIBS, 14 (10): 400-403 (1989); Benner, TIB Tech, 1 2: 158-163 (1994); Ibba and Hennecke, Bio / technology, 12: 678-682 (1994), all of which are hereby incorporated by reference at least for materials for amino acid analogs).

Molecules that resemble peptides but are not linked via natural peptide bonds can be made. For example, the linking to the amino acid or amino acid analog, the following may be _{mentioned: CH 2 NH -, - CH} 2 S -, - CH 2 --CH 2 -, - CH = CH - ( cis and _{trans), - COCH 2 -, -} CH (OH) CH 2 - and --CH H ₂ SO- (these and others, Spatola A.F., Chemis
try and Biochemistry of Amino Acids Peptides and Proteins Edited by Weinstein, Marcel Dekker New York p. 267 (1983); Spatola A. et al. F. , Vega Data Vol. 1, Issue 3, Peptide Backbone Modifications (Comprehensive Review) (March 1983); Morley Trends Pharm Sci pp. 463-468 (1980); Hudson, D .; _{Al, Int J Pept Prot Res 14:} 177-185 (1979), (- CH 2 NH -, CH 2 CH 2 -); Spatola et al, Life Sci 38: 1243-1249 (1986 ) (- CHH _2- S); Hann J. et al. Chem. Soc Perkin Trans. I307-314 (1982) (--CH--CH--, cis and trans isomers); Almquist et al., Med. Chem. _{23: 1392-1398 (1980) (-} COCH 2 -); Jennings-White et al., Tetrahedron Lett 23: 2533 (1982 ) (- COCH 2 -); Szelke et al., European Patent Application No. EP 45665 CA ( 1982): 97: 39405 (1982 ) (- CH (OH) CH 2 -); Holladay et al, Tetrahedron. Lett 24: 4401-4404 (1983) ( - C (OH) CH 2 -); and Hruby Life Sci 31: 189-199 (1982 ), (- CH 2 --S--); each of Is incorporated herein by reference. A particularly preferred non-peptide linkage is _- CH 2 NH-. It is understood that peptide analogs (eg, b-alanine, g-aminobutyric acid, etc.) can have more than one bond between the bonded atoms.

  Amino acid and peptide analogs often have the desired properties (eg, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, potency, etc.), specificity Changes (eg, widespread biological activity), reduced antigenicity, etc.).

  D-amino acids can be used to make more stable peptides. This is because D amino acids are not recognized by peptidases and the like. Systematically replacing one or more amino acids of the consensus sequence with the same type of D-amino acid (eg, substituting D-lysine at the L-lysine position) creates a more stable peptide. Can be used to Cysteine residues can be used to cyclize or to join two or more peptides together. This can be beneficial for constraining the peptide to a specific structure (Rizo and Giersch Ann. Rev. Biochem. 61: 387 (1992), which is incorporated herein by reference).

(C. Treatment method and prevention method)
(1. Inflammation)
The present invention provides a method of treating inflammation in a subject, comprising administering to the subject an effective amount of ghrelin. Inflammation can be associated with many different diseases and disorders. Examples of inflammation include, but are not limited to, inflammation associated with hepatitis, inflammation associated with the lungs, inflammation associated with burns, and inflammation associated with infectious processes. Inflammation can also be associated with liver toxicity, which in turn can be associated with cancer therapy (eg, apoptosis induction or chemotherapy, or a combination of the two).

  Inhibition of the NFκB pathway has been identified as one of the major mediators of the protective effect of ghrelin (Example 8). The NFκB regulatory genes that are regulated by ghrelin have been identified as TRCP, TOM1, AP2, GAB1 and TANK. Accordingly, a method of treating inflammation is disclosed that includes targeting TRCP, TOM1, AP2, GAB1 and TANK with ghrelin.

  Inflammation can be associated with inflammatory diseases. Examples of inflammatory diseases include asthma, systemic lupus erythematosus, rheumatoid arthritis, reactive arthritis, spondyloarthritis, systemic vasculitis, insulin-dependent diabetes mellitus, multiple sclerosis, experimental allergic encephalomyelitis, Sjögren Syndrome, graft-versus-host disease, inflammatory bowel disease (including Crohn's disease, ulcerative colitis), and scleroderma. Inflammatory diseases include autoimmune diseases (eg myasthenia gravis), Giant-Barre disease, primary biliary cirrhosis, hepatitis, hemolytic anemia, uveitis, Graves' disease, pernicious anemia, thrombocytopenia, Hashimoto's thyroiditis, ovitis, orchitis, adrenal disease, antiphospholipid syndrome, Wegener's granulomatosis, Behcet's disease, polymyositis, dermatomyositis, multiple sclerosis, vitiligo, ankylosing spondylitis, pemphigus vulgaris, Examples include psoriasis, herpes zoster, Addison's disease, Goodpasture's syndrome, Basedeau disease, thrombocytopenia, allergy, and cardiomyopathy.

Inflammation can also be associated with cancer. Examples of cancer types include lymphoma (Hodgkin and non-Hodgkin), B cell lymphoma, T cell lymphoma, leukemia (eg, myeloid leukemia and other types of leukemia), mycosis fungoides, cancer, adenocarcinoma, sarcoma , glioma, glioblastoma, neuroblastoma, plasmacytoma, histiocytoma, melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphoma or AIDS-related sarcoma, metastatic cancer, bladder carcinoma , Brain cancer, nervous system cancer, squamous cell carcinoma of head and neck, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, oral, throat, pharynx and lung squamous cell carcinoma, Colon cancer, cervical cancer, breast cancer, cervical cancer, epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, hematopoietic cancer, testicular cancer, colorectal cancer, prostate Cancers, as well as pancreatic cancers are included, but are not limited to these.

  Activated cells at the site of inflammation can also be treated. “Activated cells” are defined as cells involved in the inflammatory response. Examples of such cells include T and B cells, macrophages, NK cells, mast cells, eosinophils, neutrophils, Kupffer cells, antigen presenting cells, and vascular endothelial cells. It is not limited.

(2. Infection)
Inflammation can be caused by an infectious process in a subject. If inflammation is associated with an infectious process, this infectious process can be associated with a viral infection. Examples of viral infections include type 1 herpes simplex virus, type 2 herpes simplex virus, cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, human herpes virus 6, human herpes virus 7, human herpes virus 8, pressure ulcer Virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus genus, coronavirus genus, influenza virus A, influenza virus B, Measles virus, polyomavirus genus, human papilloma virus, RS virus, adenovirus, coxsackie virus, dengue virus, mumps virus, poliovirus, rabies virus, rous sarcoma virus, yellow fever virus, ebola virus Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Mary Valley encephalitis virus, West Nile encephalitis virus, Rift Valley fever virus, Rotavirus A group, Rotavirus B group, Rotavirus C group Sindbis virus, simian immunodeficiency virus, human T cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1, and human immunodeficiency virus type 2 For example, but not limited to.

If the inflammation is associated with an infectious process, the infectious process can be associated with a bacterial infection. The bacterial infection process can be caused by either gram positive or gram negative bacteria. Gram positive bacteria can be selected from the group consisting of: tuberculosis, M. et al. bovis, M.M. typhimurium, M. et al. bovis strain BCG, BCG substrain, M. avium, M.M. intracellulare, M. et al. africanum, M.M. Kansasii, M.M. marinum, M.M. ulcerans, M.M. avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcus agalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillus anthracis, B. subtilis, Nocardia asteroides and other Nocardia species, Streptococcus viridans group, Peptococcus species, Peptostreptococcus species, Actinomyces
israelii and other Actinomyces species, and Propionibacterium acnes.

Gram negative bacteria may be selected from the group consisting of: Clostridium tetani, Clostridium perfringens, Clostridium botulinum, other Clostridium species, Pseudomonas aeruginosa, other Pseudomonas species, Campylobacter species, Vibrio cholerae, Ehrlichia species, Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurella multicida, other Pasteurella species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Sal monella species, Shigella species Brucella abortus, other Brucella species, Chlamydi trachomatis, Chlamydia psittaci, Coxiella burnetti, Escherichia coli, Neiserria meningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Yersinia pestis, Yersinia enterolitica, other Yersinia species, Escherichia coli, E. coli. hirae and other Escherichia species, as well as other Enterobacteriacae, Brucella abortus and other Brucella species, Burkholderia cepacia, Burkholderia pseudomallei, Francisella tularensis, Bacteroides fragilis, Fusobascterium nucleatum, Provetella species and Cowdria ruminantium.

  The gram positive and gram negative bacteria in the above example are not intended to be limiting, but are representative of a larger population including all gram positive and gram negative bacteria and gram test non-responsive bacteria Intended to be. Examples of other species of bacteria include but are not limited to: Abiotrophia, Achromobacter, Acidaminococcus, Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes, Alloiococcus , Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum, Anaerohabdus, Arachnia, Arcanobacterium, Arcobacter, Arthrob cter, Atopobium, Aureobacterium, Bacteroides, Balneatrix, Bartonella, Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia, Bordetella, Brachyspira, Brevibacillus, Brevibacterium, Brevundimonas, Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium, Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea , Cellulomonas, Ce ntipeda, Chlamydia, Chlamydophila, Chromobacterium, Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella, Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia, Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister, Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella, Ehrlichia, Eikenella, Empedobacte , Enterobacter, Enterococcus, Erwinia, Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium, Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella, Fusobacterium, Gardnerella, Gemella, Globicatella, Gordona, Haemophilus, Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella, Kingella, Klebsiella, Kocuria, Koserella, Ku rthia, Kytococcus, Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella, Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria, Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus, Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella, Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis, Ochrobactrum, eskovia, Oligella, Orientia, Paenibacillus, Pantoea, Parachlamydia, Pasteurella, Pediococcus, Peptococcus, Peptostreptococcus, Photobacterium, Photorhabdus, Plesiomonas, Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia, Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella, Ralstonia, Rhodococcus, Rickettsia Rocharimaea R seomonas, Rothia, Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella, Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum, Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus, Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella, Tatumella, Tissierella, Trabulsiella, Treponema, Tropherima, Tsa amurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio, Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella.

  If inflammation is associated with an infectious process, this infectious process can be associated with a parasitic infection. Examples of parasitic infections include, but are not limited to: Toxoplasma gondii, Plasmodium species (eg, Plasmodium falciparum, Plasmodium vivax, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, Plasmodium species, (E.g. Leishmania major), Schistosoma (e.g. Schistosoma mannoni and other Shistosoma species), and Entamoeba histolytica.

  If the inflammation is associated with an infectious process, the infectious process can be associated with a fungal infection. Examples of fungal infections include but are not limited to: Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicillium marneffi, and Alternaria alternata.

(3. Sepsis)
In addition, infection can be associated with sepsis. Sepsis, also known as systemic inflammatory response syndrome (SIRS), is a serious disease caused by intensifying bloodstream infections by toxin-producing bacteria. Sepsis occurs in 2 out of every 100 hospitalized patients. This sepsis is caused by a bacterial infection and can start anywhere in the body. Common sites include the kidney (upper urinary tract infection), liver or gallbladder, intestine (usually seen with peritonitis), skin (cellulitis), and lung (bacterial pneumonia). It is not limited.

  LPS-induced endotoxemia in mice is a well-recognized model for inducing septic shock and is also associated with anorexia due to excessive production of pro-inflammatory mediators. Despite the majority of data, the cause of systemic inflammatory response syndrome (SIRS) remains unknown and various therapeutic approaches have yielded minimally beneficial results (Riedemann et al., J. Clin Invest. 112: 460-467 (2003), Luheshi et al., Proc. Natl. Acad. Sci. USA 96: 7047-7052 (1999)). Although LPS acts directly on mononuclear cells, the resulting endotoxemia also affects a wide variety of cells and tissues and is associated with refractory catabolic states.

  Ghrelin infusion in LPS challenged mice significantly inhibited pro-inflammatory cytokines IL-1α and IL-1β, IL-6 and TNF-α in the circulation and lymph nodes between liver, spleen, lung and intestinal membrane Prove to lead to. Furthermore, LPS-induced endotoxemia results in inhibition of ghrelin secretion (Hataya et al., Endocrinology. 144: 5365-5371 (2003)) and ghrelin infusion increases body weight in septic animals (Murray et al. Gastroenterology. 125: 1492-1502 (2003)). Thus, inhibition of ghrelin secretion following LPS challenge exacerbates ongoing inflammatory damage and promotes catabolic progression. Furthermore, LPS-induced inflammatory anorexia was also demonstrated to be significantly reduced in ghrelin-treated mice. Thus, inclusion bodies of ghrelin and synthetic GHS are candidates for the treatment of SIRS. Ghrelin also plays a regulatory role in chronic conditions (eg, Helicobacter pylori infection), where persistent gastrointestinal disease is associated with lower levels of ghrelin (49) Correction leads to upregulation of ghrelin secretion.

  Meningitis can also accompany sepsis. In children, sepsis is associated with bone infection (osteomyelitis). In hospitalized patients, common sites of infection include veins, surgical wounds, surgical drains, and skin failure sites known as decubitus ulcers or tears. Infection is often confirmed by positive blood cultures, which can be negative in individuals taking antibiotics. In sepsis, lowering blood pressure produces a shock. Major organs and tissues, including kidney, liver, lung and central nervous system, stop functioning normally. Sepsis is often life threatening, especially in people with a weakened immune system or other medical illness.

(4. Transplantation)
Inflammation can be associated with transplant rejection in a transplant or implant recipient. As disclosed above, “transplant rejection” is defined as an immune response elicited by the presence of extraneous blood or tissue in a subject's body. In one example of transplant rejection, antibodies are formed against foreign antigens in the transplanted material. The transplant is, for example, a tissue transplant, a cell transplant, or an organ transplant (eg, liver, kidney, skin, cornea, pancreas, pancreatic islet cell, eye, heart, or any other organ that can be transplanted in vivo) obtain.

  Transplant immunology refers to a wide sequence of events that occur after an allograft or xenograft has been removed and then transplanted into a recipient. Tissue is damaged at both the graft site and the transplant site. An inflammatory response follows immediately and activates the biochemical cascade. Such inflammatory responses can be mitigated using the methods taught herein. In an inflammatory response, antigen is recognized and a series of specific or non-specific cellular responses occur. Causes unrelated to tissue damage antigens (ie, ischemia, hypothermia, reperfusion injury) are the result of mechanical trauma when the graft is retrieved and blood supply disruption. In contrast, antigen-dependent causes of tissue damage include immune-mediated damage.

  Macrophage-releasing cytokines (eg, tumor necrosis factor, interleukin-1), which increase the intensity of inflammation by stimulating the inflammatory endothelial response; these endothelial changes cause many T cells at the site of transplantation Help to replenish.

  Damaged tissue releases pro-inflammatory mediators such as Hageman factor (factor XII), which triggers several biochemical cascades. The coagulation cascade induces fibrin and several related fibrinopeptides, which promote local vascular permeability and attract neutrophils and macrophages. The kinin cascade mainly produces bradykinin, which promotes vasodilation, smooth muscle contraction, and increased vascular permeability.

  Rejection is the result of the recipient's autoimmune response to non-self antigens expressed by the donor tissue. In hyperacute rejection, transplant subjects are serologically presensitized to alloantigens (ie, graft antigens are recognized as non-self). Histologically, polymorphonuclear leukocytes (PMN) are present in the graft vasculature and are associated with extensive microthrombin formation and platelet accumulation. There is little or no leukocyte infiltration. Hyperacute rejection manifests within minutes to hours of transplantation. Hyperacute rejection has become relatively rare since the introduction of graft recipient routine pre-transplant screening for anti-donor antibodies.

  In acute rejection, graft antigens are recognized by T cells; the resulting cytokine release ultimately leads to tissue distortion, insufficient vascularity, and cell destruction. Histologically, leukocytes are present and dominated by an equivalent number of macrophages and T cells. These processes can occur within 24 hours of implantation and can occur over a period of days to weeks.

  In chronic rejection, pathological tissue remodeling results from periplant and post-transplant trauma. Cytokines and tissue growth factors induce smooth muscle cell proliferation, induce migration, and induce the production of new matrix substances. Interstitial fibroblasts are also induced to produce collagen. Histologically, enhanced neointimal formation occurs in the aorta and medium vessels and to a lesser extent in the veins of the graft. Leukocyte infiltration is usually mild or not even present. All of these result in decreased blood flow, followed by regional tissue ischemia, fibrosis, and cell death (Prescilla et al., The website of emedicine, Immunology of Transplant Rejection, updated June 20, 2003. ).

  Transplant rejection is within 1-10 minutes of transplantation, or within 10 minutes-1 hour of transplantation, or within 1-10 hours of transplantation, or within 10-24 hours, within 24-48 hours of transplantation, transplantation Within 48 hours to 1 month, 1 month to 1 year of transplantation, 1 year to 5 years of transplantation, or longer after transplantation.

  Any animal that is subject to inflammation can be treated by this method. Thus, the subject can be any mammal, preferably a human, and this subject includes mice, rats, cows, guinea pigs, hamsters, rabbits, cats, dogs, goats, sheep, monkeys, Horses and chimpanzees can be mentioned, but are not limited to these.

(5. Loss of appetite)
The present invention provides a method of treating anorexia nervosa in a subject by administering to the subject an effective amount of ghrelin. Anorexia can be caused by a wide variety of substances, diseases and disorders. Examples of such include, but are not limited to: emotional upset, nervousness, loneliness, tension, anxiety, bereavement, depression, anorexia nervosa, nervous cachexia syndrome, acute and Chronic infection (as above), HIV, pregnancy, cancer, arteriosclerosis, inflammation (both acute and chronic, and mild inflammation), hyperthyroidism, pharmaceuticals and street drugs, chemotherapeutic agents, amphetamines, sympathetic nerves Agents (including ephedrine), antibiotics, cough and cold preparations, codeine, morphine, demerol, and digitalis. As shown in Example 7, ghrelin treatment significantly attenuated LPS-induced anorexia and resulted in increased appetite in non-LPS treated mice.

  Mild inflammation can be associated with aging, which is associated with an increase in inflammatory cytokines, including IL-6. Increased inflammatory mediator with age is associated with “anorexia of aging” and frailty (Ershler, WB and Keller, TE 2000. Age-associated increased interleukin-6 gene expression, late life diseases, and frailty. Annu. Rev. Med. 51: 245-270). Ghrelin supplementation treatment for weak and aging subjects can reduce progressive inflammatory disorders and promote improved food intake and anabolic processes.

(6. Cytokines)
Also disclosed is a method of inhibiting cytokine secretion, the method comprising administering an effective amount of ghrelin. For example, cytokines can be inhibited at the site of inflammation. The cytokine can be expressed by a cell selected from the group consisting of T cells, B cells, dendritic cells, and mononuclear cells.

  Examples of cytokines and immunomodulators that can be used in the methods of the invention include those associated with humoral inflammation (eg, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 9, IL-10, IL-13 and transforming growth factor-β (TGF-β)), and those that contribute to cellular inflammation (eg, IL-1, IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, IL-12, interferon (IFN), IFN-γ inducer (IGIF), TGF-β, and TNF-α and TNF-β). It is not limited. In accordance with the present invention, ghrelin can be used to modulate cytokines and / or immune modulators to both treat acute episodes of disease and / or maintain the state of a subject in a non-inflammatory situation obtain.

  Cytokines are proteins produced by cells that affect the behavior of other cells. Cytokines produced by lymphocytes are often called lymphokines or interleukins (IL). Cytokines act on specific cytokine receptors on the cells that they affect. Binding to the receptor induces activity (eg, proliferation, differentiation, or death) in the cell. Several cytokines, particularly IL-1, TNF-α, IL-6, IL-11, IL-8 and other chemokines, GCSF, and GM-CSF play an important role in mediating acute inflammatory responses . Among these, IL-1 (α and β) and TNF are very potent inflammatory molecules: they mediate acute inflammation induced in animals by subcutaneous injection of bacterial lipopolysaccharides, It is a major cytokine and two of the major mediators of sepsis.

  Chronic inflammation develops after acute inflammation and can last for weeks or months, in some cases years. During this state of inflammation, cytokine interactions result in chemotaxis of mononuclear cells to the site of inflammation, and then macrophage activating factor (MAF) (eg, IFN-γ, MCP-1 and others) in place. ) Activate macrophages, whereas migration inhibitory factors (MIF) (eg, GM-CSF and IFN-γ) retain macrophages at their inflammatory sites. Macrophages synthesize long-term low levels of IL-1 and TNF, which are responsible for some of the resulting clinical symptoms (eg, anorexia, cachexia, fever, sleepiness, and leukocytosis) Contributes to the inflammatory process. Cytokines known to mediate chronic inflammatory processes are those associated with humoral inflammation (eg, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-13 and transforming growth factor-β (TGF-β)), and those that contribute to cellular inflammation (eg, IL-1, IL-2, IL-3, IL-4, IL- 7, IL-9, IL-10, IL-12, interferon (IFN), IFN-γ inducer (IGIF), TGF-β, and TNF-α and TNF-β) (Feghali et al., Frontiers in Bioscience 2, d12-26 (January 1, 1997)).

  Pro-inflammatory cytokine production by cells of the innate immune system plays an important role in mediating the initial host defense against invading pathogens. Furthermore, the inability to control the nature or duration of the host inflammatory response can often mediate adverse host effects, such as those found in chronic inflammatory diseases. For example, in the early stages of sepsis, the host inflammatory response is in a superactive state with a dominant increase in the production of proinflammatory cytokines, which mediate host tissue damage and fatal shock. It is considered. Thus, the ability of the innate immune system to direct production levels of pro- and anti-inflammatory cytokines is important in limiting or regulating the nature of the host's inflammatory response.

  Production of inflammatory cytokines by the immune system, particularly leukocytes, plays an important role in the development of anorexia-cachexia syndrome (Hart et al. (1988), Kotler et al. (2000), Ershler et al. (2000)). Examples of cytokines that are considered to be associated with inflammatory anorexia include IL-1β, IL-6, and TNF-α. Peripherally administered ghrelin has been shown herein to block IL-1β-induced anorexia and produces a positive energy balance by promoting food intake and reducing energy expenditure. The inhibitory effect of ghrelin on the expression of pro-inflammatory cytokines indicates a regulatory role for ghrelin and GHS-R in the control of cytokine-induced anorexia. Furthermore, the combination with IL-1β and leptin has also been shown to inhibit the expression of ghrelin in the stomach (Cohen, J Nature 420: 885-891 (2003)) and gastric ghrelin expression is Increased in deletion mice. Leptin and ghrelin are considered to exhibit interrelated antagonistic effects on food intake at the hypothalamic level (Nakazato et al. (2001), Inui, A (2001)), leptin is a member of the gp130 family of cytokines, Induces a strong Th1 response (Hosoda et al., J. Biol. Chem. 278: 64-70 (2003)) and is regarded as a pro-inflammatory inducer (Loffreda, S. et al., FASEB J.12). : 57-65 (1998); Zarkesh-Esfahani et al., J. Immunol.167: 4593-4599 (2001), Lord et al., Nature.394: 897-901 (1998), Hosoda et al., J. Biol. : 64 70 (2003), Dixit et al., Endocrinology.144: 5595-5603 (2003)). The effect of leptin on dietary intake is controlled in part by an increase in IL-1β levels in the hypothalamus (Janik et al., J. Clin. Endocrinol. Metab. 82: 3084-3086 (1997)). Similarly, anorexic effects of IL-1 are mediated through increased leptin levels (Lambert et al., Proc. Natl. Acad. Sci. USA. 98: 4652-4657 (2001)).

It has been demonstrated that leptin can directly induce IL-1β, IL-6 and TNF-α mRNA expression and secretion by human T cells and PBMC. Leptin and several other gp130 ligands, including LIF, CNTF, and IL-5, exhibit similar effects on host metabolism (Beretta et al., Peptides. 23: 975-984 (2002), Wallenius). Et al., Nature Med. 8: 75-79 (2002)). Furthermore, IL-6 ^{− / −} deficient mice develop obesity in a manner similar to leptin deficient mice (Laviano et al., Nutrition. 18: 100-105 (2002)). Although leptin has been shown to be associated with cachexia, the level of leptin is not elevated in many cancer-related epilepsy states (Doehner et al., Eur. J. Endocrinol. 145: 727-). 735 (2001)), this is most likely due to a systemic decline in adipose tissue. However, cachexia found in patients with chronic heart failure is associated with hyperleptinemia (Nagaya, N. et al. Circulation. 104: 1430-1435 (2001)). In contrast, ghrelin reduces cachexia associated with chronic heart failure in rats (Van den Berghe et al., J. Clin. Endocrinol. Metab. 84: 1311-1323 (1999)). And the GHS-R analog, GHRP-2, counteracts protein catabolism, skeletal muscle proteolysis, and osteoporosis in severely ill patients with a state of depression (Sanna et al., J. Clin. Invest. 111). : 241-250 (2003)). Furthermore, increased leptin circulating levels in the mouse multiple sclerosis (MS) model modulate inflammatory anorexia and disease susceptibility (Sun, Y. et al., Mol. Cell. Biol. 23: 7973-7981 ( 2003)). Starvation-induced suppression of leptin levels dramatically reduced the development of EAE in this model (Sun, Y. et al. (2003)). Fasting is not only associated with a decrease in serum leptin levels and a strong increase in circulating ghrelin levels (Cummings et al. (2002), Inui, A. (2001)), but also observed fasting resistance in the MS mouse model. Inflammatory effects are also mediated by ghrelin. Given that starvation control is most important for species survival, the complex circuitry of the compensatory mechanism has evolved protection against the deletion of one or more of these regulators.

  Ghrelin function not only controls cytokine expression induced by activation, but also functions as a regulatory signal against the virus in the immune system that controls leptin-induced expression of these same inflammatory regulators. The mutual regulatory effects of these hormones on the expression of IL-1β, IL-6 and TNF-α by immune cells has a wide range of implications for the development of epilepsy disease, aging and frailty. Proposed interventions to reduce ghrelin levels or block GHS-R for the treatment of obesity can cause progressive inflammatory injury potentiation or lead to immune degeneracy obtain. In contrast, the novel anti-inflammatory effect of ghrelin within the immune system has advantages in addressing a wide range of inflammatory conditions and anorexia-cachexia syndrome associated with cancer.

(7. Treatment)
The factors and methods disclosed herein are beneficial to subjects facing or at risk of inflammation, and subjects facing anorexia. Since the factors and methods disclosed herein reduce the severity or duration of inflammation, any subject who can benefit from the reduction of inflammation may use the methods and methods disclosed herein. Can be treated by factors.

  A composition comprising an agent disclosed herein in a pharmaceutically acceptable carrier can be orally, parenterally (eg, intravenously), by intramuscular injection, by intraperitoneal injection, transdermally. In particular, it can be administered externally, locally, etc., but local intranasal administration or administration by inhalation is typically preferred. As used herein, “topical intranasal administration” means delivery of the composition into the nose and intranasal via one or both nostrils, and a nucleic acid and vector spray mechanism or fluid. It can include delivery by a drop mechanism or delivery via aerosol. The latter delivery can be effective when multiple animals are to be treated simultaneously. Administration of the composition by inhalation can be through the nose or mouth via delivery by a spray mechanism or a droplet mechanism. Delivery can also be direct to any area of the respiratory system (eg, lungs) via intubation. The exact amount of composition required will depend on the species, age, weight and general condition of the subject, the severity of the disorder being treated, the particular nucleic acid or vector used, the mode of administration, etc. , Varies from subject to subject. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

  Parenteral administration of the composition, when used, is generally characterized by injection. Injectables can be prepared either in conventional form, liquid solutions or suspensions, solid forms suitable for solution by suspension in liquid prior to injection, or as emulsions. Most recently improved parenteral administration approaches include the use of slow or sustained release systems such that a constant dose is maintained. See, for example, US Pat. No. 3,610,795, which is hereby incorporated by reference in its entirety with respect to the method taught.

  The composition can be in solution or in suspension (eg, taken up by microcapsules, liposomes or cells). These compositions can be targeted to specific cells via antibodies, receptors, or receptor ligands. The following references are examples of using this technology to target specific proteins to a given tissue (Senter et al., Bioconjugate Chem. 2: 447-451 (1991); Bagshawe, KD). , Br. J., Cancer 60: 275-281 (1989); Bagshawe et al., Br. J. Cancer 58: 700-703 (1988); Senter et al., Bioconjugate Chem. 4: 3-9 (1993); Cancer Immunol. Immunother. 35: 421-425 (1992); Pietersz and McKenzie, Immunolog. Reviews 129: 57-80 (1992); and Roffler et al., Bioch. m. Pharmacol 42: 2062-2065 (1991)): vehicles such as “stealth” and other antibody-conjugated liposomes, including liquid-mediated drug targeting to colon cancer, cell specific ligands Receptor-mediated DNA targeting, tumor targeting directed to lymphocytes, and highly specific retroviral targeting to mouse glioma cells in vivo. In general, receptors are involved in pathways of endocytosis, either constitutively or ligand-induced. These receptors cluster in clathrin-coated pits, enter cells via clathrin-coated vesicles, and acidified endosomes, where the receptors are sorted And then recycled to the cell surface, stored intracellularly, or degraded in lysosomes. This internalization pathway has various functions (eg nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor level control). To help. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and intracellular mechanisms of cell-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10: 6, 399-409 (1991)).

(A. Pharmaceutically acceptable carrier)
Administration of ghrelin or a fragment thereof disclosed herein can occur in combination with other therapeutic agents. Thus, the agents of the present invention can be administered alone or in combination with one or more therapeutic agents. For example, a subject can be treated with the disclosed agents alone, or chemotherapeutic agents, antibodies, antiviral agents, steroidal and non-steroidal anti-inflammatory agents, conventional immunotherapeutic agents, cytokines, chemokines And / or in combination with growth factors. Combinations can be in bulk (eg, as a mixture), simultaneously but separately (eg, via separate venous lines to the same subject), or sequentially (eg, of a compound or drug) One may be administered first, followed by the second). Thus, the term “combination” or “combined” is used to refer to two or more agents, either in a batch, simultaneous or sequential administration.

  The drug delivery disclosed herein can be used in therapeutic combination with a pharmaceutically acceptable carrier. Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administration of drugs to humans and include solutions such as sterile water, saline, and solutions buffered to physiological pH. These compositions are administered intramuscularly or subcutaneously. Other compounds are administered according to standard procedures used by those skilled in the art.

  Pharmaceutical compositions can include carriers, thickeners, diluents, buffers, preservatives, surface-activating agents and the like in addition to the molecule of choice. One or more components (eg, a pharmaceutical composition may also include antibacterial agents, anti-inflammatory agents, anesthetics, etc.).

  The pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical, oral, by inhalation, or parenteral, for example, by intravenous infusion, subcutaneous injection, intraperitoneal injection, or intramuscular injection. The disclosed compounds can be administered parenterally, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (eg olive oil), and injectable organic esters (eg ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or non-volatile oil. Intravenous vehicles include fluid and nutrient replenishers, electrolyte supplements (eg, Ringer's dextrose based supplements, etc.) preservatives and other additives (eg, antibacterial agents, Antioxidants, chelating agents, and inert gases may also be present .
Preparations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, water, powder or oily bases, thickeners and the like may be essential or desirable.

  Compositions for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids, or binders may be desirable.

  Some of the compositions can potentially be administered as pharmaceutically acceptable acid or base addition salts. These salts are inorganic acids (eg hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid and phosphoric acid) and organic acids (eg formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, Formed by reaction with oxalic acid, malonic acid, succinic acid, maleic acid and fumaric acid) or inorganic bases (eg sodium hydroxide, ammonium hydroxide, potassium hydroxide) and organic bases (eg monoalkyl Amines, dialkylamines, trialkylamines and arylamines, and substituted ethanolamines).

(B. Dose)
The substances of the invention can be delivered in effective amounts or concentrations. An effective concentration or amount of a substance is one that results in treatment or prevention in an inflammatory response or a decrease in appetite. One of skill in the art knows how to determine an effective concentration or amount according to methods known in the art and provided herein. One skilled in the art can use in vitro assays to optimize the in vivo dosage of a particular substance, including the concentration and time course of administration.

  The dosage range for administration of the substance is a range that is sufficiently large to produce the desired effect upon which the symptoms of the disorder are affected. For example, the dosage range is, for example, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg per kg body weight. It can be 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20 mg, or any amount in between. In particular, the amount of ghrelin that can be administered can be about 0.5 mg / kg body weight or 1-15 mg / kg body weight.

  The dosage should be such that it does not cause side effects (eg, undesired cross-reactions, anaphylactic reactions, etc.). In general, dosage will vary according to the age, condition, sex and degree of disease in the patient and can be determined by one skilled in the art. Dosage may be adjusted by the individual physician in the event of any combination contraindications. The dosage can vary and can be administered for one day or several days with one or more daily doses.

  For example, the following studies can be conducted to evaluate the efficacy of treatment of humans with disorders characterized by inflammation using substances that modulate the activity of cytokines. Patients who have failed standard medical treatment (including prednisone and / or immunomodulators known in the art) for the control of the disorder (eg, pulmonary Patients with active inflammation) can be selected. Drug efficacy can be monitored. Patients can be randomized into two different protocols. In one protocol, the subject may still receive the first drug therapy, and in the second protocol, the subject will receive the drug therapy after receiving a substance that modulates cytokine activity (eg, ghrelin). Can be weakened gradually.

  In one example, ghrelin can be infused over a 2 hour period until inflammation or anorexia symptoms subside, or a dose of about 0.5 mg per kg body weight is infused over a 2 hour period each time Can be done. The subject's blood pressure, heart rate and body temperature can be monitored before and during the 2 hour infusion at 30 minute intervals. The subject can also receive routine inflammation monitoring.

  As noted above, the agents disclosed herein can be administered with other forms of therapeutics. For example, the molecule can be administered with antibodies, antibiotics, or other cancer treatment protocols as described above, or viral vectors. When the agent (factor) is present in the vector as described above, the vector containing the nucleic acid for therapeutic purposes may also contain ghrelin or a fragment thereof.

(C. Nucleic acid approach for delivery)
A substance of the invention, including ghrelin, can also be administered to a patient or subject in vivo and / or ex vivo as a nucleic acid preparation (eg, DNA or RNA) encoding the substance (eg, ghrelin) As a result, the patient's own cells or the subject's own cells take up the nucleic acid and produce and secrete the encoded substance.

  The nucleic acid of the invention can be present in the form of naked DNA or RNA, or the nucleic acid can be present in a vector to deliver the nucleic acid to a cell. This allows the DNA fragment to be under the transcriptional control of a promoter, as will be appreciated by those skilled in the art. The vector can be a commercially available preparation such as, for example, an adenoviral vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada)) Delivery of this nucleic acid or vector to cells can be via various mechanisms. As delivery, commercially available liposome preparations (eg, LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM Bio (Promega Inte. , WI) and other liposomes developed according to standard procedures in the art. The nucleic acids or vectors of the present invention can be delivered in vivo by electroporation, in this regard, available from Genetronics, Inc. (San Diego, Calif.) And SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ) ).

As an example, delivery of the vector can be via a viral system (eg, a retroviral vector system capable of packaging a recombinant viral genome) (eg, Pastan et al., Proc. Natl. Acad. Sci. USA). 85: 4486, 1988; Miller et al., Mol. Cell. Biol. 6: 2895, (1986)). The recombinant retrovirus can then be used to infect nucleic acids encoding the broad neutralizing antibodies (or active fragments thereof) of the invention, thereby delivering to the infected cells. The precise method of introducing the modified nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure, including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5: 941-948, (994)), the use of adeno-associated virus (AAV) vectors ( Goodman et al., Blood 8 4: 1492-1500 (1994)), use of lentiviral vectors (Naidini et al., Science 272: 263-267 (1996)), and use of pseudoretroviral vectors (Agrawal et al., Expert. Hematol. 24: 738-747 (1996)). Physical transfer techniques can also be used, such as, for example, liposome delivery and receptor-mediated mechanisms and other endocytosis mechanisms (for example, Schwartzenberger et al., Blood 87: 472-478 to name a few. , (1996 ) ). The present invention can be used in combination with any of these methods or other commonly used gene transfer methods.

By way of example, when the nucleic acid encoding an antibody of the invention is delivered to a subject's cells in an adenoviral vector, the dosage for administration of adenovirus to a human is 10 ⁷ to 10 ⁹ plaque forming units per injection. (Pfu), but can be as high as 10 ¹² pfu per injection (Crystal, Hum. Gene Ther. 8: 985-1001 (1997); Alvarez and Curiel, Hum. Gene Ther. 8: 597-613). (1997)). The subject may receive a bolus injection or if further injection is required, the subject will be at an interval of 6 months (or at other appropriate intervals as determined by the skilled person). It can be repeated for an indefinite period and / or until the efficacy of the treatment is established.

  Parenteral administration of the nucleic acids or vectors of the invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquids or suspensions, as solids suitable for liquids suspended in liquids prior to injection, or as emulsions. More recently, an improved approach for parenteral administration involves the use of delayed release or sustained release systems so that a constant dosage is maintained. See, for example, US Pat. No. 3,610,795, which is incorporated herein by reference. For a discussion of suitable formulations and various routes of administration of therapeutic compounds, see, eg, Remington: The Science and Practice of Pharmacy (19th edition) A.A. R. See Gennaro, Mack Publishing Company, Easton, PA (1995).

  The following examples are described to provide those skilled in the art with the methods by which the compounds, compositions, articles, devices and / or methods claimed in the rewrite are made and evaluated. Are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in degrees Celsius (° C.) or ambient temperature, and pressure is at or near atmospheric.

(Example 1: General method)
Human subjects Ferresis packs were prepared from 6 healthy male donors between the ages of 22 and 37 for PBMC and T cell isolation.

  (Mice) Male 10 to 22 g BALB / c mice (Taconic, Germantown, New York) 8-10 weeks old were used. With regard to the care of the National Research Council's Life Sciences Laboratory Animal Resource Committee, the animal's distress and stress were minimized in accordance with the guidelines proposed by the committee. Each animal was fed a laboratory rodent diet and had free access to water.

  LPS-induced inflammation Endotoxin shock in mice was described as previously described (Bochkov et al., 2002. Nature. 419: 77-8), 10 μg LPS (E. coli serotype 055: B5, Sigma). Was induced by intraperitoneal injection (ip). The animals were also treated with i.p. of ghrelin in PBS 24 hours and 30 minutes before LPS administration. p. A single injection (5 mg / kg body weight) was made. Mice were sacrificed 4 hours and 24 hours after LPS challenge and organs and serum were collected.

T cell isolation and culture Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque density centrifugation. T cells were purified from PBMC using a human T cell enrichment column (R & D systems) via high affinity negative selection according to the manufacturer's instructions. Flow analysis typically revealed a purity greater than 90%. Plates were used for anti-human CD3 antibody (BD Pharmingen, San Diego, Calif.) (200 ng / ml) at a concentration of 3 × 10 ⁶ cells / ml in AIM-V serum-free medium for 24 hours. I was stimulated.

Immunofluorescence staining Cell staining was performed as previously described (17). Briefly, cells were human anti-GHS-R goat IgG, anti-GHS-R rabbit IgG (recognizing 186-202 amino acids near the C-terminus of human GHS-R) (Santa Cruz Biotech, Santa Cruz, California) , Incubated overnight at 4 ° C. with different combinations of anti-ghrelin rabbit IgG, anti-pro-ghrelin precursor rabbit IgG (Phoenix peptides, Belmont, Calif.). Lipid rafts were visualized using cholera toxin-Alexa fluoro (AF) 594 (Molecular Probes, Eugene, Oregon) at 20 μg / ml for 45 minutes. The Golgi apparatus was stained using goat anti-mouse Golgin-97 (Golgi marker) (Molecular Probes, Eugene, Oregon). Thereafter, the cells, AF - 488 appropriate secondary antibody conjugated, and using the appropriate secondary antibody conjugated with AF-594 was labeled with. Nuclei were counterstained using dihydrochloric acid 4 ′, 6-diaminodino-2-phenylindole (DAPI) (1 μg / ml). Images were acquired by Spot Advanced software on a Zeiss Axiovert S100 microscope (Carl Zeiss, Thornwood, New York) for 100X objects.

Flow cytometry analysis Human PBMC (1 × 10 ⁶ ) in PBS containing heat inactivated 2% FBS was fixed and stained for using 1% paraformaldehyde. Stained for CD3, CD4, CD8 PE and CD14 PE conjugated antibodies (BD Pharmingen, San Diego, California) and incubated on ice for 30 minutes. Cells were washed with PBS and then stained for GHS-R and ghrelin, followed by a specific secondary antibody conjugated with AF-488 and analyzed on a FACScan.

Intracellular calcium kinetics Measurement of intracellular calcium release in response to ghrelin and SDF-1 was performed as previously described (Sherman-Bust et al. Cancer Cell. 4: 377-386 (2003). Was incubated in PBS containing 5 μM Fura-2 AM for 30 minutes at room temperature, then the cells were washed and then resuspended in PBS to 1 × 10 ⁶ / mL. All of the objects were placed in a continuously stirred cuvette at room temperature using an LS50B spectrophotometer (Perkin-Elmer, Wellesley, Massachusetts) and the fluorescence was λ _ex1 = 340 nm, λ _ex2 = 380 nm, λ _em = Monitored at 510 nm, these data were 340 n And it is provided as a relative ratio of the excited fluorescence in 380 nm.

(Actin polymerization) Human T cells were incubated for 20 minutes with either ghrelin (100 ng / ml) or positive control SDF-1 (100 ng / ml). Cells were then fixed and permeabilized in 2% paraformaldehyde 0.1% Triton-X100 and stained for nuclei using phalloidin AF-594 and nuclei with DAPI.

  Cytokine Evaluation IL-1β, IL-6, and TNF-α in T cell supernatants after 24 hours using a commercially available ELISA kit according to the manufacturer's (Biosource, Camarillo, California) handling protocol. evaluated. Serum, cytokines were analyzed using Bio-Plex Mouse Cytokine 18-Plex Panel according to the manufacturer's instructions (Biorad, Hercules, California).

  Real-time RT-PCR analysis RT-PCR was performed as previously described (Nagazawa et al., Adv. Immunol. 71: 211-228 (1999)). CDNA was synthesized using a single-stranded Reverse Transcription kit (Life Technologies, Gaithersburg, Maryland) using total RNA (2 μg) and oligo-dT primers according to the manufacturer's instructions. PCR was set up using SYBR Green Master Mix (Applied Biosystems), 1 μl cDNA and a final concentration of 0.3 μM gene-specific primers. Thermal cycling was performed on an Applied Biosystems GeneAmp 7700 Sequence Detector and SYBR green dye intensity was analyzed using GeneAmp 7700 SDS software. Primers for human IL-1β gene, human IL-6 gene, human TNF-α gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control were purchased from Biosource International, Camarillo, Calif., And human GHS-R 1a And human ghrelin was used as previously described (Gnanapavan et al. (2002)). Mouse IL-1β primer, mouse IL-6 primer, mouse TNF-α primer, GAPDH primer and human GHS-R 1a primer were designed using ABI prism software (PE Applied Biosystems). The PCR product of GHS-R1a amplification was quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California). Primers are available when needed. PCR products were not generated from gene versus cDNA templates.

(Statistical analysis) The results were expressed as mean ± standard error ( SEM ) . Statistical analysis was performed by one-dimensional ANOVA. Significant differences between treatment groups were determined by Student-Newman-Keuls test and statistical significance was estimated as P <0.05.

(Example 2: GHS-R is a functional receptor expressed on human T cells)
Although previous results only described mRNA expression of GHS-R in lymphoid organs, this study focused on expression and spatial localization of GHS-R protein in generated human T cells. GHS-R represented a heterogeneous subcellular morphology in dormant human T cells spreading from a half-moon, punctate or diffuse phenotype (FIG. 1a upper, FIG. 8b). In dormant human T cells, the majority of ghrelin receptors are distant from GM1 ⁺ lipid rafts (upper side of FIG. 1a). However, upon T cell activation via TCR ligation, a dramatic partial reorganization of GHS-R was observed, which implicated the polarized cap phenotype and aggregation of lipid rafts. Shown (bottom of FIG. 1a). Flow cytometric analysis showed specific staining for GHS-R in up to 30% of highly purified dormant human T cells, as shown by the use of blocking peptides (FIG. 1b). In human PBMC, expression of GHS-R in CD3 ⁺ T cells, CD3 ⁺ CD4 ⁺ T cells and CD3 ⁺ CD8 ⁺ T cells is not observed in a preferential expression manner in a subset of these immune cells . In highly purified human T cells, GHS-R expression is significantly increased upon cell activation (FIG. 1c). In the presence of the antibody-specific blocking peptide, this GHS-R labeling was almost completely removed. Furthermore, there is also a significant up-regulation of GHS-R gene expression upon T cell activation, as shown by quantitative analysis of PCR products using Agilent gene chip technology and real-time RT-PCR (FIG. 1d). ). The presence of GHS-R in lipid rafts and the specific activation of the GHS-R gene upon T cell activation indicates a role for these receptors in T cell function. A similar staining pattern for ghrelin receptor was observed in activated T cells using a secondary antibody that recognizes the proximal 186-265 amino acid residues from the C-terminal region of human GHS-R (FIG. 8a).

Seven-transmembrane GPCR ligation typically results in calcium flux from intracellular stores by production of inositol triphosphates (Kojima et al. (1999), Sherman-Bust et al. (2003)). Ghrelin has been previously shown to induce intracellular calcium release in Chinese hamster oocytes (CHO cells) transfected with GHS-R (Kojima et al. (1999)). Here, cultured human T cells were used to show a significant and specific increase in intracellular [Ca ²⁺ ] in response to both full-length ghrelin protein and ghrelin 1-18 fragment (FIG. 1e). This pretreatment with [D-Lys-3] -GHRP-6 (a highly selective GHS-R antagonist) significantly reduces ghrelin-mediated intracellular calcium release from T cells, so this ghrelin-induced Was found to be GHS-R specific (FIG. 1e). Interestingly, intracellular calcium mobilization induced by ghrelin treatment is positively bound to stromal cell-derived factor 1 (SDF-1), which binds specifically to the cell surface GPCR CXCR4 and Is the same magnitude as the intracellular calcium flux observed in response to a potential T cell chemokine ligand that signals through (Sanchez-Madrid et al., EMBO J. 18: 501-511 (1999)). ). Furthermore, in calcium fluidization, GPCR ligation often involves dramatic actin skeleton and cell surface molecule remodeling and leads to polarization and often leads to directional migration of immune cells (Taub et al. (1993), Inui, A (1999)). Here, ghrelin induced a marked increase in the characteristics of the extensive membrane structure of lamellipodium with typical polarization of F-actin in a manner very similar to SDF-1 treated cells (FIG. 1f). Taken together, these data demonstrate the presence of functional GHS-R on the surface of human T cells and mononuclear cell subsets and suggest a biological role for ghrelin and GHS-R in the immune system .

Example 3: Ghrelin mRNA and protein expressed in human mononuclear cells
Among mononuclear cells, monocytes constitute an important source of pro-inflammatory cytokines, so we will test the expression of GHS-R in monocytes. Flow cytometric analysis revealed that approximately 21% of CD14 + cells expressed GHS-R (FIG. 2a). Using immunofluorescence microscopy, dispersed GHS-R expression was detected on the cell surface of purified monocytes (FIG. 2b top) and showed that the control IgG was not specifically labeled ( 2b lower side). Similarly, GHS-R expression was observed in immature and mature monocytes derived from dendritic cells. Real-time RT-PCR analysis also showed the presence of GHS-R1a mRNA in monocytes at a level comparable to primary human T cells.

(Example 4: Ghrelin selectively inhibits pro-inflammatory cytokine expression)
The classical pro-inflammatory cytokines IL-1α, IL-1β, IL-6 and TNF-α are known to play an important role in the development of anorexia-cachexia syndrome (Inui et al. 1999. Cancer). Res. 59: 4493-4501). Anorexia-cachexia syndrome is a complex multifactorial metabolic state that is associated with metabolic changes in proteins, carbohydrates and fats, resulting in anorexia, negative energy balance, weight loss and muscle itch (Kotler et al. 2000. Ann. Internal Med. 133: 622-634). Given the important role played by pro-inflammatory cytokines in regulating metabolic activity, the ability of ghrelin to regulate the production of IL-1β, IL-6 and TNF-α by activated PBMC and T cells Tested. Human PBMCs from healthy male subjects are stimulated with a polyclonal mitogen, phytohemagglutinin (PHA) and incubated for 24 hours in the presence or absence of ghrelin and GHS-R antagonist, after which the supernatant Were collected and tested for cytokine levels. Ghrelin treatment resulted in significant inhibition of IL-1β, IL-6 and TNF-α production by PBMC at levels of ghrelin ranging from 1 ng / ml to 100 ng / ml (FIG. 3a-c); however, ghrelin treatment Was unsuccessful in altering TGF-β production by these PBMCs at any concentration tested (FIG. 3d). This effect was found to be GHS-R specific. Because the addition of GHS-R antagonists to these cultures reduced this ghrelin-mediated inhibition, and the effect of ghrelin on similar cytokine production was induced by PBMC and LPS treated monocytes stimulated by concanavalin A (ConA). It was because it was observed using. Furthermore, primary human T cells stimulated for 24 hours in the presence of ghrelin with immobilized anti-CD-3 antibody showed significant dose-dependent inhibition of IL-1β and IL-6 (FIG. 3e-). g). This measurement of lactate dehydrogenase (LDH) and cell counts using trypan blue exclusion failed to show any significant difference between control and hormone treated cells, so this ghrelin-mediated inhibition was It should also be noted that it did not depend on any of the lytic effects of this hormone on PBMC. Using real-time RT-PCR analysis, ghrelin was demonstrated to significantly inhibit IL-1β, IL-6 and TNF-α mRNA expression in all donors. This indicates a reduction in cytokine production (FIG. 3h). These results indicate that ghrelin plays a role in the transcriptional control of inflammatory cytokine expression.

Example 5: Ghrelin inhibits the expression of leptin-mediated pro-inflammatory cytokines
The mechanism of action by which leptin and ghrelin regulate the production of inflammatory cytokines was determined. Observed the spatial localization of the Ob-R protein in human T cells (FIG. 4a) and protein expression and mRNA of IL-1β (FIG. 4b), IL-6 (FIG. 4c) and TNF-α (FIG. 4d) We have also demonstrated that leptin directly induces a significant dose-dependent increase in expression by primary human T cells (FIG. 4e) and a significant dose-dependent increase by PBMC. When adding ghrelin to these cultures synergistically, we observed dose-dependent inhibition of leptin-induced cytokine protein expression and cytokine gene expression by T cells in response to various concentrations of ghrelin (FIG. 4b-e). This indicates that ghrelin and leptin have a similar effect in the hypothalamus on food intake, but interact with each other to exert antagonistic effects on the expression of inflammatory cytokines in the immune system. Thus, changes in circulating levels of leptin and ghrelin have a significant effect on the production of various cytokines by immune cell populations. Such reciprocal immunomodulatory effects are important in maintaining immune cell homeostasis and thus prevent abnormal cytokine production that causes or amplifies disease and pathology.

Example 6: Human T cells express ghrelin and actively secrete this ghrelin)
Ghrelin was thought to be produced exclusively by the stomach and subsequently secreted into the peripheral circulation (Kojima et al., 1999. Nature. 402: 656-660). However, peripheral ghrelin levels have been demonstrated to increase gradually after gastrectomy, indicating that an additional cellular source of ghrelin compensates for ghrelin from the stomach (Dixit et al., 2003. Endocrinology.144: 5595-5603).

Lymphocytes are known to produce many well-characterized hormones such as GH (Hosoda et al., 2003. J. Biol. Chem. 278: 64-70). This GH exerts many autocrine and paracrine effects on the immune system (Taub et al., 1994. J. Clin. Invest. 94: 293-300). Given the potential effect of ghrelin on cytokine expression, the possibility of the presence of ghrelin endogenously produced by immune cells is postulated. The presence of immunoreactive ghrelin and GHS-R is demonstrated herein in dormant T cells with a broadly distributed phenotype (FIG. 5a top) and from a co-localized region, a ligand for ghrelin in T cells. -Shows the role of receptor interactions and autocrine. Upon linking TCR, a clear change in the spatial localization of endogenous immunoreactive ghrelin is observed, which results in a polarization of the expression phenotype. Ghrelin appears to associate specifically within GM1 ⁺ lipid rafts (middle of FIG. 5). This indicates that upon activation, ghrelin is produced and specifically targeted to lipid rafts and their own specific receptors. With further support for ghrelin synthesis by human T cells, the 117 amino acid pre-pro form of ghrelin was also co-expressed and found to be co-localized within the Golgi apparatus (bottom of FIG. 5a). Within this Golgi apparatus, pre-progrelin is probably cleaved prior to secretion and processed into its mature form. Both ghrelin and pre-proghrelin in primary T cells were canceled upon addition of antibody-specific blocking peptide (FIG. 8d).

These findings are further supported by flow cytometric analysis of various T cell subsets for mature ghrelin protein, where the majority of T cells appear to be ghrelin positive and the CD3 ⁺ CD4 ⁺ T cell portion There is no preferential expression in the assembly nor in the CD3 ⁺ CD8 ⁺ T cell subset (FIG. 5b). In addition to the expression of intracellular ghrelin by T cells, TCR ligation of these cells results in substantial levels of ghrelin protein that are secreted into the culture supernatant, which peaks at 48 hours and then decreases. (Figure 5b). Furthermore, T cell activation induced more than 5-fold ghrelin mRNA expression as demonstrated by real-time RT-PCR analysis (FIG. 5c).

  Given the presence and secretion of ghrelin by T cells, the concentration of ghrelin in the local microenvironment is not subject to the classical dilution effect that is typically seen during release from the stomach into the peripheral circulation. In addition, significantly higher levels can be reached. Thus, T cell-derived ghrelin may serve an important role in controlling cell function within the immune microenvironment or within the immune organ. Given the specific antagonistic effect of ghrelin on leptin-mediated inflammatory cytokine expression, the possible reciprocal effect of leptin on ghrelin and GHS-R expression in T cells was tested. Leptin failed to exert any significant effect on ghrelin protein production or ghrelin gene expression in human T cell cultures (FIG. 5d). Furthermore, leptin treatment resulted in a significant increase in GHS-R mRNA expression by human T cells as measured by real-time RT-PCR (FIG. 5e). Thus, down-regulation of leptin-induced cytokine expression by ghrelin builds a reciprocal regulatory signaling pathway whereby these hormones control each other's activity within the immune system (Fig. 5f). Furthermore, comparison of real-time PCR analysis of ghrelin expression in human stomach and lymphoid organs revealed that the stomach has 11 times higher expression of ghrelin than T cells, spleen and thymus (FIG. 5g). Lymphoid organs and small intestine expressed ghrelin mRNA levels 5 times higher compared to placenta.

Example 7: Ghrelin down-regulates inflammatory cytokine expression and anorexia in response to endotoxin challenge
Bacterial lipopolysaccharide (LPS) is a major component in the pathogenesis of endotoxin shock, which acts primarily on monocytes and elicits an acute phase type response in vivo to produce IL-1β, IL-6 and TNF. -Overproduction of α occurs. The amplification of these proximal cytokines has a wide range of pro-inflammatory and anorexic effects (Kotler et al., 2000. Ann. Internal Med. 133: 622-634), which is associated with sepsis and multiple organs Contributes to the etiology of failure (Cohen et al., 2003. Nature 420: 885-891; Riedemann et al., 2003. J. Clin. Invest. 112: 460-467). In an attempt to test the ability of ghrelin to modulate the in vivo expression of inflammatory cytokines, mice were treated with ghrelin before and after administration of LPS. As shown in FIG. 6, ghrelin exerted a potent anti-inflammatory effect against LPS-induced endotoxemia by inhibiting in vivo expression of IL-1β, IL-6 and TNF-α. Real-time PCR analysis of mRNA from the spleen and liver of these endotoxin-treated mice revealed strong expression of these cytokine genes 4 hours after LPS administration (FIG. 6a-c) and mRNA up to 24 hours. A significant disappearance of expression (FIGS. 6d-f) was revealed. Mice treated with ghrelin and challenged with endotoxin demonstrated attenuation of IL-1β and IL-6 mRNA expression in both spleen and liver after 4 and 24 hours (FIGS. A-f). A decrease in TNF-α mRNA was observed in both spleen and liver at 4 hours (FIG. 6c), and TNF-α expression was inhibited in the liver 24 hours after LPS administration, and there was no change in the spleen Remained (FIG. 6f). Similar inhibition of pro-inflammatory cytokines was observed in the pulmonary and intermesenteric lymph nodes of ghrelin-treated mice 4-24 hours after LPS challenge.

To measure circulating serum cytokine levels, mice were treated with LPS treatment, ghrelin treatment either 4 or 24 hours after LPS. Analysis of serum cytokines showed a significant change in circulating TNF-α after 4 hours of ghrelin treatment (FIG. 7c), but also changes in IL-1β (FIG. 7a) and IL-6 (FIG. 7b) However, significant inhibition of IL-1β and IL-6 was observed 24 hours after LPS challenge (FIGS. 7d, 7e). Serum levels of TNF-α were undetectable 24 hours after LPS challenge. To test the effect of ghrelin on endotoxin-induced anorexia, food intake was also evaluated 24 hours after administration of ghrelin and / or LPS. LPS challenged mice showed a dramatic reduction in food consumption compared to sham mice (80%), and prior ghrelin treatment resulted in a significant reduction in LPS-induced anorexia (FIG. 7f). As expected, control mice were ghrelin treated without L PS challenge also demonstrated as compared to the pseudo control a significant increase in food intake (30%). Serum IL-1β and IL-1α levels were also significantly inhibited in these mice injected with ghrelin alone when compared to sham control mice (FIGS. 7g, 7h). And serum ILα levels were inhibited 24 hours after LPS and ghrelin treatment (FIG. 7h).

Example 8: Overall gene expression profile associated with the protective effect of ghrelin in LPS-induced mouse endotoxemia
Endotoxin shock was induced in mice by intraperitoneal (ip) injection of 10 μg LPS (E. coli serotype 055: B5). The animals also received a single intraperitoneal injection of ghrelin in PBS (0.5 m / kg) 24 hours and 30 minutes prior to administration of LPS. Mice were sacrificed 4 and 24 hours after LPS challenge, and internal organs and serum were collected. RNA was extracted from the spleens of sham and treated mice and utilized for gene arrays using NIA mouse 17K arrays. Hybridization spots on the microarray filter were analyzed using array pro software and then the average intensity of the image was determined. The various intensities of each spot were normalized across the filter, and relatively overexpressed and relatively underexpressed genes between various conditions were determined with an average 1.5-fold rate of change.

  (Results) Gene expression was compared between the following conditions: 1) sham vs. ghrelin; 2) LPS (4 hours) vs. LPS (4 hours) + ghrelin; and 3) LPS (24 hours) vs. LPS (24 hours) ) + Ghrelin.

  Pseudo-vs. Ghrelin 71 known genes up-regulated and 51 ESTs up-regulated in the ghrelin gene array were compared to the mock gene array. Among the anti-inflammatory target genes, both PACAP receptor and CD22 were up-regulated. There were a total of 134 known genes that were down-regulated and 94 ESTs that were down-regulated. Anti-inflammatory target genes include lipoprotein lipase, fatty acid synthase, S-adenosyl homocysteine hydrolase, peripheral benzodiazepine receptor, TANK, serum glucocorticoid kinase (dinase) (SGK), and lysophospholipase 1 .

(LPS 4 hours vs. LPS 4 hours + ghrelin) Endotoxemia mice treated with ghrelin were measured for gene expression at 4 hours and endotoxemia at 4 hours not treated with ghrelin. Compared to diseased mice. Known genes and 76 amino upregulated by ES T is 72 amino upregulated in total was present in ghrelin array. Among the anti-inflammatory target genes, IGF-1, estrogen receptor 1 (α), and TIMP4 were upregulated. There were a total of 156 known genes that were down-regulated, and there were 95 down-regulated ESTs. Among the anti-inflammatory target genes, calmodulin 1, thioredoxin reductase, glutamate cysteine ligase, carbonic anhydrase 2, squalene epoxidase, GAB1 and Trcp were down-regulated.

  (LPS 24 hours vs. LPS 24 hours + ghrelin) In endotoxemic mice treated with ghrelin, gene expression at 24 hours was measured and compared to endotoxemic mice not treated with ghrelin. There were a total of 143 known genes that were upregulated and 118 ESTs that were upregulated. Among the anti-inflammatory target genes, Tom1 (Myb1 target), thioredoxin reductase, glutamate-cysteine ligase (catalytic subunit), and glucocorticoid-inducible gene 1 were detected. There were a total of 168 known genes that were down-regulated and 187 ESTs that were down-regulated. Among the anti-inflammatory target genes, leukotriene A4 hydrolase, PECAM, LPS-induced TN factor, TANK and Star were all detected.

  Overall, in the endotoxemia model, inhibition of the NFκB pathway was identified as one of the major mediators of the protective effects of ghrelin. NFκB regulatory genes regulated by ghrelin were identified as TRCP, TOM1, AP2, GAB1 and TANK.

(Example 9: Ghrelin levels are reduced in subjects with ulcerative colitis and Crohn's disease)
Serum ghrelin levels are reduced in patients with Crohn's disease (Figure 11). In a study conducted in 15 subjects with Crohn's disease and 13 subjects without it, ghrelin levels were found to be significantly lower in patients with Crohn's disease. Ghrelin expression is also significantly less in subjects with ulcerative colitis (Figure 12).

FIG. 1 (af) shows functional GHS-R expression in human T cells. (A) Primary human T cells were labeled for GHS-R and subcellular localization in lipid rafts was visualized in dormant cells and anti-CD-3 activated cells. (B) Flow cytometric analysis of GHS-R of highly purified human dormant T cells. Specific T cell labeling is counteracted in the presence of antibody specific blocking peptides. T cells stained with control IgG showed no specific labeling. (C) Flow analysis of GHS-R expression in highly purified (> 96%) activated CD3 ⁺ human T cells. Staining specificity was demonstrated through the use of antibody specific blocking peptides. (D) GHS-R mRNA is up-regulated upon T cell activation when evaluated using Agilent gene chip quantification and real-time RT-PCR , values are mean ± standard error ( ^* P <0.05 ). (E) Ghrelin induces intracellular calcium mobilization in cultured human T cells. T cells were stimulated with ghrelin (100 ng / ml) or SDF-1 (100 ng / ml) for 60 seconds. T cells were stimulated with GHS-R antagonist [D-Lys-3] -GHRP-6 (10 ⁻⁴ M) for 60 seconds, followed by ghrelin (100 ng / ml) for 180 seconds. (F) Ghrelin causes actin polymerization in human T cells. Cells were treated with ghrelin (100 ng / ml) and positive control SDF-1 (100 ng / ml) for 20 minutes and stained with phalloidin AF-594 for F-actin. FIG. 2 (a-b) shows that the ghrelin receptor is expressed in human monocytes. (A) Human PBMC were double-stained using CD14PE and GHS-R AF-488. (B) Immunofluorescent labeling revealed GHS-R expression on the cell surface of purified monocytes (upper panel), and the negative control failed to show any specific staining. Figure 3 (a- d) indicates that ghrelin inhibits expression of inflammatory cytokines from human PBMC and T cells. 24 hours in the presence or absence of various doses of ghrelin (closed circle) and simultaneously with GHS-R antagonist [D-Lys-3] -GHRP-6 (10 ⁻⁴ M; open circle) Human PBMC (n = 6) was stimulated by PHA (1 μg / ml) (ah) and T cells were activated via immobilized anti-CD3 antibody (eh). The collected supernatants were subsequently assayed for IL-1β (a, e), IL-6 (b, f) and TNF-α (c, g) and TGF-β (d). These cytokine protein data are expressed as mean ± standard error and are representative of 6 healthy adult donors ( ^* P <0.05). (H) Change fold of IL-1β, IL-6 and TNF-α. MRNA in T cells after normalization by GAPDH was measured by real-time RT-PCR. FIG. 4 (a-e) shows that ghrelin shows a leptin-induced increase in the inflammatory cytokine. (A) Localization of leptin receptor on the surface of human T cells (Ob-R). (Bd) Anti-CD3 mAb-activated T cells from human adult donors (n = 6) are incubated with various concentrations of leptin or at the same time biologically optimal concentrations with various concentrations of ghrelin Of leptin (100 nM). Cytokine production and mRNA expression were assessed after 24 hours in culture. The cytokines tested were (b) IL-1β, (c) IL-6, and (d) TNF-α. (E) Fold change of IL-1β, IL-6 and TNF-α. MRNA expression measured by real-time RT-PCR after normalization by GAPDH. Values are expressed as mean ± standard error ( ^* P <0.05). FIG. 5 (ag) shows that ghrelin is expressed and secreted from human T cells. (A) Co-expression of ghrelin and GHS-R in dormant T cells (top); activated T cells show that ghrelin coexists in strong GM1 ⁺ lipid rafts (middle); pre-pro-ghrelin is activated Coexist in the Golgi apparatus of human T cells (lower side). (B) Kinetics of ghrelin secretion from anti-CD3 mAb stimulated T cells. (C) Fold change in ghrelin mRNA levels upon T cell activation as assessed by real-time RT-PCR. Values are expressed as mean ± standard error ( ^* P <0.05). (D) Ghrelin expression was quantified after 24 hours in culture in T cells stimulated in the presence of immobilized anti-CD3 antibody and in the presence or absence of different concentrations of leptin. Fold change (closed bar) in expression of ghrelin mRNA as measured by real-time RT-PCR after normalization by GAPDH. Production of ghrelin protein was determined by EIA (open bar). (E) Change fold of GHS-R gene expression after normalization by GAPDH (n = 6). Values are expressed as mean ± standard error ( ^* P <0.05). (F) A hypothetical model for the functional role of ghrelin as a signal linking the immune-endocrine system in the control of food intake. (G) Comparative ghrelin mRNA expression in the stomach compared to lymphoid organs. Figure 6 (af) shows inhibition of inflammatory cytokine expression and anorexia in a mouse endotoxemia model. Real-time PCR analysis for inflammatory cytokine mRNA in spleen and liver 4 hours (4h) and 24 hours (24h) after administration of LPS and ghrelin in BALB / c mice. Ct values for cytokines were normalized by GAPDH and expressed as fold change (n = 6) over the Ct values of the sacrificed control mimics. At 4 and 24 hours after LPS injection, ghrelin inhibited IL-1β (a, d) and IL-6 (b, e) transcription in both spleen and liver. In the spleen, although TNF-alpha onset current is attenuated after 4 hours of injection of LPS, ghrelin could not further inhibit TNF-alpha mRNA in 24 hours in the spleen. However, ghrelin continued to significantly suppress TNF-α mRNA in the liver (c, f). FIG. 7 (ai) shows cytokine levels in the serum of treated mice after treatment with LPS and ghrelin. The cytokines tested were IL-1β (a), IL-6 (b), and TNF-α (c) at 4 hours, and IL-1β (d) and IL-6 (e) at 24 hours. . Ghrelin stimulates food intake in LPS challenged mice (f). Ghrelin treatment inhibits basal IL-1β and ILα secretion in the periphery (g, h). Ghrelin also inhibits serum IL-1α levels 24 hours after LPS challenge (i). Values are expressed as mean ± standard error ( ^* P <0.05). FIG. 8 ( ad ) shows GHS-R expression. (A) GHS-R expression in activated purified human T cells, which utilizes an antibody that recognizes 186-265 amino acids in the region near the C-terminus of the GHS-R peptide of human origin. (B) Differential pattern of GHS-R expression in dormant human T cells. The lower panel represents punctate GHS-R expression in dormant T cells and shows somewhat less coexistence with GM-1 positive rafts. (C) Proliferation of human T cells in response to anti-CD3 mAb and ghrelin treatment (open circles) and IL-2 levels (painted circles). Ghrelin had no significant effect (p <0.05) on either T cell proliferation or IL-2 secretion. (D) Specificity of anti-ghrelin labeling and anti-pre-proghrelin labeling in purified T cells. These images were obtained using the same exposure time and the same gain. FIG. 9 shows that total ghrelin and acylated ghrelin are co-expressed in human PBMC. (A) anti-rabbit antibody, followed by using a secondary antibody conjugated with AF-594 (red), total ghrelin was labeled. (B) Expression of acylated ghrelin was assessed using an anti-guinea pig antibody and a specific secondary antibody conjugated with AF-488 (green). Nuclei were stained with DAPI. (C) Overlapping shows that about 30% of cells expressing total ghrelin also simultaneously express the active octanoylated form of ghrelin. FIG. 10 shows that T-cell derived ghrelin is important for the control of prostatic cytokines and pro-inflammatory chemokine homeostasis (ab). Ghrelin expression in T cells was down-regulated using siRNA. Reduction of ghrelin levels increases pro-inflammatory cytokines in human T cells. FIG. 11 shows a decrease in ghrelin levels in patients with Crohn's disease. FIG. 12 shows a decrease in ghrelin expression in ulcerative colitis.

Claims (16)

A composition for treating inflammation in a subject, said composition comprising an effective amount of
a ) a fragment of SEQ ID NO: 1; or
b ) a fragment of SEQ ID NO: 2 ,
A composition comprising one or more of the following. The composition of claim 1, wherein the inflammation is associated with an infectious process, liver toxicity, or inflammatory disease. The composition of claim 2, wherein the liver toxicity is associated with cancer treatment. A composition for treating anorexia in a subject, the composition, the effective amount,
a) a fragment of SEQ ID NO: 1; or
b) a fragment of SEQ ID NO: 2,
A composition comprising one or more of the following. 5. The composition of claim 4, wherein the anorexia is caused by atherosclerosis, inflammation, aging, or a psychological disorder. 6. The composition of claim 5, wherein the psychological disorder is anorexia-cachexia syndrome. A composition for treating sepsis in a subject, said composition comprising an effective amount ,
a) a fragment of SEQ ID NO: 1; or
b) a fragment of SEQ ID NO: 2,
A composition comprising one or more of the following. 8. The composition of claim 7, wherein the sepsis is endotoxemia. 1 mg to 50 mg per kg body weight of the subject ,
a) a fragment of SEQ ID NO: 1; or
b) a fragment of SEQ ID NO: 2,
The composition according to claim 7, comprising one or more of the following. 1 mg to 50 mg per kg body weight of the subject ,
a) a fragment of SEQ ID NO: 1; or
b) a fragment of SEQ ID NO: 2,
The composition according to claim 7, comprising one or more of the following. About 5 mg / kg body weight of the subject ,
a) a fragment of SEQ ID NO: 1; or
b) a fragment of SEQ ID NO: 2,
The composition according to claim 7, comprising one or more of the following. A composition for inhibiting cytokine secretion, the composition comprising an effective amount ,
a) a fragment of SEQ ID NO: 1; or
b) a fragment of SEQ ID NO: 2,
A composition comprising one or more of the following. 13. The composition of claim 12, wherein the cytokine is inhibited at the site of inflammation. 13. The composition of claim 12, wherein the cytokine is selected from the group consisting of IL-1, IL-6, TNF- [alpha], INF- [gamma], IL-12 and p40. 13. The composition of claim 12, wherein the cytokine is expressed by a cell selected from the group consisting of T cells, B cells, dendritic cells, and mononuclear cells. The composition of claim 2, wherein the inflammatory process is a viral infection, a bacterial infection, a parasitic infection, or a fungal infection.